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A MANUAL 



OF 



CLINICAL DIAGNOSIS 

BY MEANS OF MICEOSCOPIC AND 
CHEMICAL METHODS 



STUDENTS, HOSPITAL PHYSICIANS, AND PRACTITIONERS 



BY 

CHARLES E. SIMON, B.A., M.I). 

FORMERLY OF THE RESIDENT STAFF OF THE JOHNS HOPKINS HOSPITAL J PROFESSOR OF 

CLINICAL PATHOLOGY AT THE BALTIMORE MEDICAL COLLEGE J CLINICAL 

MICROSCOPIST TO THE UNION PROTESTANT INFIRMARY. 



SIXTH EDITION, THOROUGHLY REVISED 



ILLUSTRATED WITH 177 ENGRAVINGS AND 24 PLATES IN COLORS 




LEA BROTHERS & CO. 
PHILADELPHIA AND NEW YORK 



\^ 



LIBRARY of CONGRESS [ 
Two Crates Received 
JUL 15 190? 
Coj>yriefit Entry 

IkStf CL XXC, No. 

/? /? 6~o 



COPY fci. 



U~«J 



Entered according to Act of Congress, in the year 1907, by 

LEA BROTHERS & CO. 

in the Office of the Librarian of Congress. All rights reserved. 



M Y Wl F E 

WHO HAS SO FAITHFULLY AIDED IN ITS PREPARATION 

THIS EDITION ALSO 

IS 

AFFECTIONATELY DEDICATED 



PEEFACE TO SIXTH EDITIOX 



In preparing the sixth edition of the Clinical Diagnosis the author 
was confronted with an important problem. A great deal of new mate- 
rial had to be introduced, but the size of the volume, which had steadily 
grown within the ten years of its existence, could not be exceeded. 
It was accordingly necessary to go oyer the entire work carefully and 
to cut out even*thing that was not of clearly practical value, to con- 
dense, and to rewrite. The amount of labor involved was consider- 
able, but the object has been, it is hoped, satisfactorily achieved.. 

The chapter on the Blood has been further enlarged and brought 
thoroughly to date. Every page in the work has undergone a radical 
review. A new chapter on the Opsonins has been introduced, in which 
the subject-matter has been conservatively and, it is hoped, fairly pre- 
sented; full details are given regarding the technical portion of the 
subject, in which the writer's experience as a pioneer worker may 
prove of value. 

Two appendices have been added. The first deals with the prepa- 
ration of culture media, and may prove of service to teachers who use 
the book not only as a text-book of clinical diagnosis, but also as a guide 
to the student's work in bacteriology. The second represents an out- 
line of a course in clinical laboratory methods, and is presented at the 
request of teachers in clinical microscopy in many of our medical 
schools, in which the subject is steadily growing in importance. The 
"course" is based upon the work which the writer has conducted for 
post-graduates during the past ten years in his own laboratory, and 
is designed to be thoroughly practical and comprehensive. 

Numerous illustrations in black and white, as well as a number of 
colored plates, mostly from the brush of Airs. Simon, have been added. 

The author wishes to thank the medical profession for the continued 
favorable reception of the Diagnosis, the pioneer work in America, 
and trusts that the present edition also will serve its purpose as a trust- 
worthy guide to the medical student, general practitioner, and the 
laboratory research worker. 

Charles E. Simon. 
1302 Madison Ave., Baltimore, 
1907. 



CONTENTS. 



CHAPTER I. 

THE BLOOD. 

PAGE 

General considerations 17 

General characteristics of the blood . 17 

color 17 

odor 18 

specific gravity 18 

determination according to Hammerschlag 18 

determination according to Schmaltz and Peiper 19 

indirect estimation of the hemoglobin 19 

reaction . 20 

estimation of the alkalinity according to Lowy 21 

estimation of the alkalinity according to Engel 21 

estimation of the alkalinity according to Dare 22 

Chemical examination of the blood 25 

general chemistry of the blood 25 

coagulation of the blood 27 

blood pigments . 29 

hemoglobin and oxyhemoglobin 29 

hemoglobinemia 33 

carbon monoxide hemoglobin 34 

nitric oxide hemoglobin 35 

hydrogen sulphide hemoglobin 35 

carbon dioxide hemoglobin 35 

hematin 35 

hemin 36 

methemoglobin 37 

hematoidin 38 

hematoporphyrin 38 

the proteids of the blood 39 

the carbohydrates 40 

sugar . 40 

estimation of the sugar in the blood 41 

Williamson's diabetic blood test . . 42 

glycogen 42 

cellulose 42 

urea 42 

uremia . 43 

ammonia 43 

uric acid and xanthin bases 43 

fat and fatty acids 45 

lactic acid 48 

biliary constituents 49 

acetone 50 

cholin 50 

Microscopic examination of the blood 51 

the red corpuscles 51 

variations in the size of the red corpuscles 51 

variations in the form of the red corpuscles ....... 52 



viii CONTENTS 

Microscopic examination of the blood — Continued. page 

variations in the color of the red corpuscles and color index . . 53 

variations in the number of the red corpuscles 54 

behavior toward aniline dyes (polychromatophilia) .... 60 

granular degeneration ' 61 

Cabot's ring bodies 64 

Ehrlich's hemoglobinemic Innenkorper 65 

nucleated red corpuscles 65 

normoblasts 65 

megaloblasts 67 

the leukocytes 69 

general differentiation of the various forms of leukocytes ... 69 
differentiation of the leukocytes according to their behavior toward 

aniline dyes . 70 

the small mononuclear leukocytes 70 

the large mononuclear leukocytes 73 

the polynuclear neutrophilic leukocytes 74 

the polynuclear eosinophilic leukocytes . 77 

the mast-cells 78 

the myelocytes 80 

Tiirck's irritation forms (phlogocytes) 82 

iodophilia 83 

leukocytosis 84 

polynuclear neutrophilic hyperleukocytosis 85 

polynuclear neutrophilic hypoleukocytosis ....... 97 

neutrophilic karyomorphism 101 

polynuclear eosinophilic hyperleukocytosis 102 

polynuclear eosinophilic hypoleukocytosis 108 

lymphocytosis 109 

lymphopenia 112 

variations in number of the large mononuclear leukocytes . . . 112 

variations in number of the mast-cells 113 

myelocytosis 114 

the plaques 118 

the dust particles of Muller 120 

General technique 120 

slides and cover-glasses 120 

the blood mount ........ 121 

fixation 123 

The aniline dyes and principles of staining . . . . 125 

Methods of staining 129 

the eosinate of methylene blue . . . . .129 

Ehrlich's triacid stain 130 

the Romanowsky methods . . ... . . . 132 

method of Hastings 132 

method of Leishman 134 

method of Wright 135 

method of Giemsa 136 

method of Goldhorn 137 

Demonstration of iodophilia 137 

Enumeration of the corpuscles of the blood 137 

method of Simon ' 137 

enumeration of the leukocytes 138 

enumeration of the red cells .140 

differential leukocyte counting .143 

enumeration of the plaques 144 

The hematokrit 144 

volume index 147 

Estimation of hemoglobin . 147 

Dare's hemoglobinometer . 148 

FleischPs hemoglobinometer . 150 

Gowers' hemoglobinometer (Sahli's modification) . . . . . . 152 

Talquist's method 152 



CONTENTS ix 

TAGE 

Estimation of blood iron 153 

Kryoscopic examination of the blood 154 

Osmotic resistance of the red cells 157 

Bacteriology and parasitology of the blood 158 

typhoid fever 159 

agglutination test 160 

paratyphoid fever 165 

pneumonia 166 

pyogenic bacteriemia 168 

go nococcus septicemia 171 

anthrax ... 172 

acute miliary tuberculosis 173 

glanders 174 

influenza . . . . ... . . . 174 

Malta fever 174 

bubonic plague . 175 

malaria 177 

trypanosomiasis 187 

relapsing fever 189 

typhus fever 190 

Kala-azar . 190 

syphilis 191 

spotted fever . . 191 

filariasis . f . . 191 

distomiasis (bilharziasis) 195 

anguilluliasis 196 



CHAPTER II. 

THE SECRETIONS OF THE MOUTH. 

Saliva 198 

general characteristics 198 

chemistry of the saliva 198 

microscopic examination of the saliva 199 

pathological alterations 201 

Special diseases of the mouth 202 

tuberculosis of the mouth 202 

actinomycosis 202 

catarrhal stomatitis 202 

ulcerative stomatitis 202 

gonorrheal stomatitis 202 

thrush 202 

Tartar 203 

Coating of the tongue 203 

Coating of the tonsils 203 

pharvngomvcosis leptothrica . 203 

tonsillitis 204 

Vincent's angina 204 

diphtheria 205 

scarlatina 208 

glandular fever 208 



CHAPTER III. 

THE GASTRIC JUICE AND THE GASTRIC CONTENTS. 

The secretion of gastric juice 209 

Test meals 210 

the test breakfast of Ewald and Boas 210 



x CONTENTS 

Tests meals — Continued. page 

the test breakfast of Boas 210 

the test dinner of Riegel 210 

the double test meal of Salzer 210 

The stomach tube . ' 211 

contra-indications to the use of the tube . 211 

introduction of the tube 211 

General characteristics of the gastric juice 213 

amount 213 

Chemical examination of the gastric juice 214 

the acidity of the gastric juice 214 

determination of the acidity of the gastric juice 215 

the amount of free hydrochloric acid 217 

euchlorhydria 218 

hypochlorhydria 218 

anachlorhydria 218 

hyperchlorhydria 218 

test for free acids 219 

test for free hydrochloric acid . . 219 

the dimethyl-amino-azo-benzol test 219 

the phloroglucin-vanillin test 220 

the resorcin test 221 

the tropeolin test " 221 

the combined hydrochloric acid 222 

quantitative estimation of the hydrochloric acid ....... 222 

Topfer's method . ". ", 222 

deficit of hydrochloric acid 223 

Sahli's method 223 

Martius and Liittke's method 224 

Leo's method . 226 

the ferments of the gastric juice and their zymogens 227 

pepsin and pepsinogen 227 

tests for pepsin and pepsinogen . . . . . . . . . 229 

quantitative estimation 229 

chymosin and chymosinogen 230 

tests for chymosin and chymosinogen 231 

quantitative estimation 232 

analysis of the products of albuminous digestion . . . . . . 232 

tests for the products of carbohydrate digestion . 233 

lactic acid 233 

mode of formation and clinical significance 233 

tests for lactic acid 235 

Kelling's test 235 

Uffelmann's test . . . • 235 

Strauss' test 236 

Vournaso's test 236 

Boas' test 237 

quantitative estimation of lactic acid according to Boas' method 230 

the fatty acids 240 

mode of formation and clinical significance 241 

tests for butyric acid 241 

tests for acetic acid 242 

quantitative estimation of the fatty acids 242 

gases 244 

acetone • • 244 

vomited material 244 

food material 246 

mucus 246 

gastrosuccorrhea mucosa 246 

saliva 246 

bile 246 

pancreatic juice 247 

blood 247 



CONTENTS xi 

Chemical examination of the gastric juice — Continued. page 

test of Miiller and Weber 247 

Donogany's method . . . . . 247 

pus 248 

stercoraceous material 248 

parasites 249 

odor . 249 

Microscopic examination of the gastric contents 249 

the Boas-Oppler bacillus 250 

sarcinae 250 

protozoa 251 

shreds of mucous membrane 251 

tumor particles 252 

Examination of the motor power of the stomach 252 

Leube's method 252 

the salol test of Ewald and Sievers 253 

Examination of the resorptive power of the stomach 253 

Indirect examination of the gastric juice . . 254 

Gimzburg's method 254 



CHAPTER IV. 

THE FECES. 

Examination of normal feces 256 

general characteristics . . . 256 

number of stools 256 

amount 256 

consistence and form 257 

odor - "-..-. 258 

color 258 

aloin test 260 

guajac test 261 

macroscopic constituents 261 

alimentary detritus 261 

mucous cylinders 262 

concretions 263 

foreign bodies 265 

microscopic constituents 265 

constituents derived from food 266 

morphological elements derived from the alimentary canal . . 270 

leukocytes 271 

blood corpuscles 271 

crystals '271 

mucus 273 

Bacteriology of the feces 273 

Bacillus dvsenterise 276 

Bacillus typhi 278 

Bacillus acidophilus 280 

Bacillus vulgaris 281 

Bacillus pyocyaneus 282 

Bacillus coli communis 282 

Bacillus lactis aerogenes 283 

Bacillus cholerse 283 

Bacillus tuberculosis 285 

Animal parasitology 286 

protozoa 286 

Entamoeba dvsenterise 286 

Entamoeba coli 289 

Paramceba hominis - 289 

Cercomonas hominis 290 

Trichomonas intestinalis 291 

Megastoma entericum 292 



xii CONTENTS 

Animal parasitology — Continued. page 

Balantidium coli 293 

vermes 295 

Taenia saginata 295 

Taenia solium - 296 

Taenia nana . . . . 297 

Taenia diminuta 299 

Taenia cucumerina 299 

Taenia Africana 299 

Taenia Madagascariensis 299 

Bothriocephalic latus . . 300 

Krabbea grandis 302 

Distoma hepaticum " 302 

Distoma lanceolatum . 302 

Distoma Buskii 304 

Distoma sibiricum 304 

Distoma spatulatum 304 

Distoma conjunctum 304 

Distoma heterophyes 304 

Amphistomum hominis 304 

Ascaris lumbricoides 305 

Ascaris mystax 307 

Ascaris maritima 307 

Oxyuris vermicularis 307 

Uncinaria duodenalis ' . 307 

Trichocephalus hominis 311 

Trichina spiralis 311 

Strongyloides intestinalis 312 

Chemistry of the feces 316 

reaction . . 316 

chemical composition 317 

phenol, indol, skatol 318 

fatty acids 319 

cholesterin 319 

biliary acids . 320 

pigments 320 

purin bodies 322 

mucin 322 

albumin 323 

albumoses 323 

carbohydrates 323 

ptomains 323 

Meconium 323 



CHAPTER V. 

THE NASAL SECRETION. 

Physiology and pathology of the nasal secretion 325 

CHAPTER VI. 

THE SPUTUM. 

General technique 326 

General characteristics of the sputa 327 

amount 327 

consistence 327 

color 328 

odor 328 

specific gravity 329 

configuration of sputa 329 



CONTENTS 



Xlll 



PAGE 

Macroscopic constituents of sputum 330 

cheesy particles 330 

elastic tissue 330 

fibrinous casts 330 

Curschmann's spirals : 332 

echinococcus membranes 333 

concretions . 333 

foreign bodies 333 

Microscopic examination 334 

leukocytes 334 

red blood corpuscles 335 

epithelial cells 336 

elastic tissue 337 

animal parasites . . 339 

Entamoeba dysenterise 339 

Trichomonades . 339 

Cercomonades 339 

Taenia echinococcus 339 

Distoma pulmonale 343 

Distoma hematobium 343 

vegetable parasites . 344 

the tubercle bacillus 344 

methods of staining 345 

Gabett's method. . , . . . . . . . . 345 

Weigert-Ehrlich's method 346 

Ziehl-Neelsen's method 346 

cultivation of tubercle bacillus 347 

the Diplococcus pneumoniae 348 

the bacillus of influenza . 349 

the bacillus of whooping-cough . . . . . . . . 350 

the smegma bacillus 351 

the typhoid bacillus . . . 351 

the plague bacillus : 351 

Micrococcus catarrhalis 351 

Micrococcus tetragenus 351 

staphylococci and streptococci 351 

streptothrices . . 351 

actinomycosis 351 

blastomycetes 353 

moulds 353 

Oidium albicans . 354 

Sarcina pulmonalis 354 

crystals '354 

Charcot-Leyden crystals 354 

hematoidin 356 

cholesterin 356 

fatty acid crystals 356 

leucin and tyrosin 357 

calcium oxalate 357 

triple phosphates 357 

the pneumoconioses 357 

anthracosis 357 

siderosis 358 

chalicosis . 358 

stycosis 358 

Chemistry of the sputum 358 

CHAPTER VII. 

THE URINE. 

General characteristics of the urine 360 

general appearance ... . 360 

color 361 



xiv / CONTENTS 

General characteristics of the urine — Continued. page 

odor 362 

consistence 362 

quantity , 362 

polyuria 363 

oliguria 365 

specific gravity 366 

determination of the specific gravity . 367 

determination of the solid constituents 368 

Reaction 369 

determination of the acidity of the urine 371 

Chemistry of the urine 373 

general chemical composition of the urine 373 

quantitative estimation of the mineral ash of the urine 373 

the chlorides 374 

test for chlorides in the urine 377 

quantitative estimation of the chlorides by the method of Salkow- 

ski-Volhard 377 

direct method 381 

the phosphates ... 382 

test for the phosphates in the urine 386 

quantitative estimation of the total amount of phosphates . . 387 

separate estimation of the earthy and alkaline phosphates 390 

removal of the phosphates from the urine 390 

the sulphates . 390 

test for the sulphates in the urine 394 

quantitative estimation of the sulphates 394 

quantitative estimation of the total sulphates 394 

quantitative estimation of the conjugate sulphates . . . 395 

neutral sulphur 396 

quantitative estimation 398 

urea 399 

properties of urea . . . 407 

urea nitrate 408 

urea oxalate 408 

quantitative estimation of urea , 409 

hypobromite method 409 

Folin's method 412 

estimation of nitrogen according to Kjeldahl 413 

ammonia 415 

quantitative estimation 416 

Folin's method 416 

uric acid 417 

properties of uric acid 423 

tests for uric acid 424 

quantitative estimation of uric acid (Folin's method) .... 424 

. xanthin bases . 427 

quantitative estimation (Salkowski's method) ....... 427 

hippuric acid 429 

properties of hippuric acid 430 

quantitative estimation of hippuric acid 431 

kreatin and kreatinin . 432 

properties of kreatin and kreatinin . 433 

test for kreatinin in the urine 434 

quantitative estimation of kreatinin in the urine (Folin's method) . 434 

oxalic acid . . 436 

properties of oxalic acid 438 

tests for oxalic acid .... 439 

quantitative estimation of oxalic acid (Dunlop's method) . . . 439 

Albumins 440 

serum albumin . . - 441 

serum globulin '. 454 

albumoses 454 



CONTENTS xv 

Albumins — Continued. page 

Bence Jones' albumin 456 

peptone 457 

hemoglobin 458 

fibrin 459 

nucleo-albumin 460 

histon and nucleohiston 461 

tests for albumin 461 

tests for serum albumin 462 

nitric acid test 462 

boiling test 464 

potassium ferrocyanide test 465 

trichloracetic acid test 465 

picric acid test . 466 

Spiegler's test . 466 

special test for serum albumin 466 

quantitative estimation of albumin 467 

old method of boiling 467 

Esbach's method 467 

gravimetric method 468 

test for serum globulin and its quantitative estimation 469 

tests for albumoses 469 

Bang's method 470 

examination for peptone (polypeptids) 470 

tests for Bence Jones' albumin 471 

tests for (mucin) nucleo-albumin 472 

tests for hemoglobin . 473 

Heller's test 473 

Donogany's test 474 

test for fibrin 474 

test for histon . . 474 

Carbohydrates 474 

glucose 474 

tests for sugar 433 

Trommer's test 483 

Fehling's test 484 

Bottger's test with Nylander's modification 484 

fermentation test 485 

phenvlhydrazin test 486 

polarimetric test 487 

quantitative estimation of sugar 489 

Fehling's method 489 

Gerrard and Allan's method 490 

differential density method 49 1 

Einhorn's method 492 

Lohnstein's method 492 

polarimetric rnethod 494 

lactose 495 

levulose 495 

maltose 495 

dextrin ...... 496 

laiose . . . ' . ... 496 

pentoses 496 

Tollens' orcin test 497 

Tollens' phloroglucin test 497 

Glucuronic acid 497 

Inosit 498 

Urinary pigments and chromogens 493 

normal pigments 493 

urochrome 499 

uroerythrin . 50O 

normal chromogens 501 

indican 501 



xvi CONTENTS 

Urinary pigments and chromogens — Continued. page 

tests for indican 503 

quantitative estimation 504 

urohematin 506 

uroroseinogen 507 

pathological pigments and chromogens ..'..'•■ 508 

blood pigments 508 

hematin 508 

urombrohematin and urofuscohematin 508 

urohematoporphyrin 508 

biliary pigments 510 

Smith's test 512 

Huppert's test 512 

Gmelin's test (as modified hy Rosenbach) . . . . .. 512 

Gmelin's test 512 

biliary acids 512 

cholesterin 513 

pathological urobilin . . . 513 

melanin and melanogen . . . . . . . . . . .516 

phenol 517 

alkapton (homogentisinic acid) 518 

blue urines 521 

green urines 521 

pigments referable to drugs . " . .521 

Ehrlich's diazo reaction 522 

benzaldehyde reaction 526 

Acetone 527 

tests for acetone 529 

Legal's test 529 

Lieben's test 529 

Frommer's test 530 

Dennige's test 530 

quantitative estimation 531 

Diacetic acid 532 

Gerhardt's test 532 

Arnold's test 533 

Oxybutyric acid 533 

estimation 534 

Crotonic acid 535 

Lactic acid . . . . . . . . 535 

Oxyamygdalic acid 536 

Volatile fatty acids 537 

Amino-acids 538 

Fat 539 

Ferments 540 

Gases 541 

Ptomains 542 

isolation of diamins 543 

Kryoscopic examination of the urine 546 

Sediments 546 

Microscopic examination of the urine 546 

non-organized sediments 549 

sediments occurring in acid urines 549 

uric acid.. 549 

amorphous urates 550 

calcium oxalate 551 

monocalcium phosphate 552 

hippuric acid 552 

calcium sulphate 553 

cystin 553 

leucin and tyrosin 554 

xanthin 556 

soaps of lime and magnesia 557 



CONTENTS xvii 

Microscopic examination of the urine — Continued. page 

bilirubin and hematoidin 557 

fat ... 557 

sediments occurring in alkaline urines 558 

basic phosphate of calcium and magnesium 558 

neutral calcium phosphate 559 

magnesium phosphate 559 

amm oniomagnesium phosphate 560 

calcium carbonate 560 

ammonium urate 560 

indigo 561 

organized constituents of urinary sediments 561 

epithelial cells 561 

leukocytes 563 

red blood corpuscles 567 

tube casts 570 

examination 571 

true casts 572 

hyaline casts 572 

brown granular casts 571 

waxy casts * 571 

pseudocasts 576 

cylindroids 576 

clinical significance of tube casts 577 

spermatozoa 579 

parasites 580 

vegetable parasites - 580 

animal parasites 586 

tumor particles 587 

foreign bodies 587 

CHAPTER VIII. 

TRANSUDATES AND EXUDATES. 

Transudates 588 

general characteristics 588 

specific gravity 588 

chemistry of transudates 590 

microscopic examination 591 

Exudates 591 

serous exudates 591 

technique 594 

bacteriological examination of 595 

inoscopy 596 

chemistrv of 597 

pus . 598 

general characteristics of pus 598 

chemistry of pus 598 

microscopic examination of pus 599 

leukocytes 599 

giant corpuscles 600 

detritus 600 

red blood corpuscles 600 

pathogenic vegetable parasites 600 

protozoa 601 

vermes 601 

crystals 602 

technique 602 

gonorrheal pus 602 

the gonococcus 603 

putrid exudates 604 

chvlous and chvloid exudates 604 



XVJ11 



CONTENTS 



Exudates — Continued . pag e 

syphilitic material 606 

Spirochete pallida 606 

CHAPTER IX. 

THE CEREBROSPINAL FLUID. 

Amount 610 

Appearance . 610 

Specific gravity 611 

Reaction 612 

Chemical composition 612 

Microscopic examination 615 

Cytology 615 

Bacteriology 616 

Toxicity . 618 

CHAPTER X. 

THE EXAMINATION OF CYSTIC CONTENTS. 

Cysts of the ovaries and their appendages 619 

Hydatid cysts 621 

Hydronephrosis 622 

Pancreatic cvsts 622 



CHAPTER XI. 

THE SEMEN. 

General characteristics 623 

Chemistry of the semen 623 

Microscopic examination of the semen 624 

Pathology of the semen 624 

The recognition of semen in stains 625 

CHAPTER XII. 

VAGINAL DISCHARGES. 

Bacteriology 627 

Vaginal blennorrhea 628 

Menstruation 649 

The lochia 649 

Vulvitis and vaginitis i . 629 

Membranous dysmenorrhea 630 

Cancer 630 

Gonorrhea 630 

Abortion 631 



CHAPTER XIII. 

THE SECRETION OF THE MAMMARY GLANDS. 

The secretion of milk in the newly born 632 

Colostrum 632 

The secretion of milk in the adult female 633 

Human milk 633 

The milk in disease 634 



CONTENTS xix 

The milk in disease — Continued. page 

determination of the specific gravity 635 

estimation of the fat 637 

estimation of lactose 637 

estimation of the proteids 637 

CHAPTER XIV. 

THE OPSONINS. 

The opsonins 639 

Technique 642 

The opsonic index 643 

APPENDIX. 

A. 

Preparation of culture media 649 

B. 

Outline of a course in clinical microscopy -. 654 

Lectures < 654 

Laboratory exercises 656 



CLINICAL DIAGNOSIS, 



CHAPTER I. 

THE BLOOD. 

GENERAL CONSIDERATIONS. 

If blood is allowed to flow directly from an artery into a vessel 
surrounded by a freezing mixture, and containing one-seventh its 
volume of a saturated- solution of sodium sulphate, or a 25 per 
cent, solution of magnesium sulphate (1 volume to 4 volumes of 
blood), it will be observed that after some time a sediment, pre- 
senting the color of arterial blood, has formed at the bottom, which 
is covered by a layer of clear, straw-colored fluid — the blood plasma. 
Upon microscopic examination the sediment will be seen to contain: 

(a) Numerous homogeneous, non-nucleated, circular, biconcave 
disks. These measure on an average 7.5 p. in diameter, and are of a 
faint greenish-yellow color when viewed through the microscope, 
while en masse they present the color of arterial blood — the erythro- 
cytes or red corpuscles of the blood. 

(b) Roundish or irregularly shaped nucleated cells which are for 
the most part granular and far less numerous than the red corpus- 
cles, and devoid of coloring matter — the leukocytes, colorless or 
white corpuscles of the blood. 

(c) Minute colorless disks, measuring less than one-half the diam- 
eter of a red corpuscle — the so-called blood plaques, or blood plates 
of Bizzozero. 

GENERAL CHARACTERISTICS OF THE BLOOD. 

Color. — Chemical examination of the blood shows that its color is 
referable to the presence of an albuminous, iron-containing substance 
— hemoglobin — in the bodies of the red corpuscles, which is character- 
ized by its great avidity for oxygen, and forms a compound therewith, 
known as oxyhemoglobin. The relatively larger amount of the 
latter encountered in the arteries, as compared with the veins, causes 
the difference in the appearance of arterial and venous blood, the 
2 



18 THE BLOOD 

former presenting a bright scarlet-red, the latter a dark-bluish color. 
A bright cherry-red color is noted in poisoning with carbon monoxide, 
while a brownish-red or chocolate color is observed in poisoning 
with potassium chlorate, anilin, hydrocyanic acid, and nitrobenzol. 
A milky appearance is frequently seen in well-marked leukemia. 
In chlorosis and hydremic conditions, as would be expected, the 
blood is pale and watery. 

Odor. — The peculiar odor of the blood, which varies in different 
animals, the halitus sanguinis of the ancients, is due to the presence of 
certain volatile fatty acids, and may be rendered more distinct by 
the addition of concentrated sulphuric acid. 

Specific Gravity. — The specific gravity of the blood in healthy 
adults varies between 1.058 and 1.062, being higher on an average 
in men, 1.059, than in women, 1.056, and children — boys 1.052, 
girls 1.050. Generally speaking, it is proportionate to the amount 
of hemoglobin and the volume of red corpuscles. It is diminished 
by fasting, the ingestion of solids and liquids, gentle exercise, preg- 
nancy, etc. It depends, moreover, upon the bloodvessel from which 
the specimen is taken, being higher in venous than in arterial blood. 

Under pathological conditions the specific gravity may vary be- 
tween 1.025 and 1.083. In nephritis, chlorosis, the anemias in 
general, and in cachectic conditions (carcinoma of the stomach, etc.) 
it may diminish to 1.031. In phthisis it is diminished in the third 
stage (1.040 to 1.042), and in the first stage (1.049) in those patients 
in whom the onset has been very gradual. In the second stage 
normal figures are obtained (1.058 to 1.060), corresponding to the 
relatively high percentage of hemoglobin (90 to 95 per cent.) which 
is then noted, and which is referable no doubt to a concentration of 
the blood. An increased specific gravity is met with in febrile 
diseases (typhoid fever, 1.057 to 1.063), conditions associated with 
pronounced cyanosis (emphysema, fatty heart, uncompensated val- 
vular disease, 1.054 to 1.068), and obstructive jaundice, 1.062. 
The highest values have been found in enterogenous cyanosis, 1 .067 
to 1.083. 

Method of Determining the Specific Gravity of the Blood. 
Hammerschlag's Method. — A carefully dried cylinder, measuring about 
10 cm. in height, is partly filled with a mixture of chloroform (sp. 
gr. 1.526) and benzol (sp. gr. 0.889), having a specific gravity of 
1.050 to 1.060. Into this solution a drop of blood is allowed to fall 
directly from the finger, pressure being avoided, and care taken 
that the drop does not come in contact with the walls of the vessel. 
The drop should not be too large, as otherwise it will separate into 
droplets, giving rise to inaccurate results. Should the drop sink 
to the bottom, it is apparent that the specific gravity of the mixture 
is lower than that of the blood, necessitating the addition of chloroform. 
This should be added drop by drop while the mixture is thoroughly 



GENERAL CHARACTERISTICS OF THE BLOOD 



19 



stirred. If, on the other hand, the drop should tend toward the surface 
it is best to add an amount of benzol sufficient to cause the blood to 
sink to the bottom, and then to bring it to the proper degree of sus- 
pension by the subsequent addition of chloroform. As soon as the 
drop remains suspended the mixture is filtered, and its specific 
gravity ascertained by means of an accurate areometer registered to 
the fourth decimal. The figure obtained is the specific gravity of 
the blood. The chloroform-benzol mixture may be kept indefinitely. 

With practice, results sufficiently accurate for clinical purposes 
may thus be obtained with an expenditure of very little time. The 
examination should in each case be made at the same hour, as the 
specific gravity undergoes diurnal variations. 

Instead of the chloroform-benzol mixture, one of chloroform and 
olive oil may be employed, as suggested by Van Spanje. It has 
the advantage of being less volatile than the other. Three parts of 
chloroform and one of oil give a mixture with a specific gravity 
of 1.056. 

Schmaltz and Peiper's Method. — Where delicate scales are avail- 
able the method of Schmaltz and Peiper may be employed. A 
capillary tube, measuring about 12 cm. in length and 1.5 mm. in 
width, with its ends tapering to a diameter of 0.75 mm., is filled with 
blood and carefully weighed. The weight of the blood, divided by 
the weight of an equivalent volume of distilled water, indicates the 
specific gravity. 

As the result of numerous investigations it may now be regarded 
as an established fact, that with the exception of nephritis, circulatory 
disturbances, leukemia, posthemorrhagic anemia, and that resulting 
from inanition, the specific gravity of the blood varies directly with 
the amount of hemoglobin and the volume of the red corpuscles. 
A simple method is thus given by means of which hemoglobin esti- 
mations can be made in the absence of more expensive instruments. 
In the following table the specific gravities, as obtained with Ham- 
merschlag's method, and that of Schmaltz and Peiper are given, 
with the corresponding amounts of hemoglobin : 



Specific gravity 




Specific gravity 




according to 


Hemoglobin. according to 


Hemoglobin. 


Hammerschlag. 




Schmaltz and Pei] 


3er. 




1.033-1.035 . 


. 25-30 per 


cent. 1.030 


20 per cent 


1.035-1.038 . 


. 30-35 


1.035 


. . 30 


" 


1.038-1.040 . 


. 35-40 


1.038 


. . 35 


a 


1.040-1.045 . 


. 40-45 


1.041 


. . 40 


a 


1.045-1.048 . 


. 45-55 


1.0425 


. . 45 


u 


1.048-1.050 . 


. 55-65 


1 0455 


. . 50 


a 


1.050-1.053 . 


. 65-70 


' 1.048 


. . 55 


" 


1.053-1.055 . 


. 70-75 


' 1.049 


. . 60 


" 


1.055-1.057 . 


. 75-85 


1.051 


. . 65 


11 


1.057-1.060 . 


. 85-95 


1.052 
1.0535 


. . 70 
. . . 75 








1.056 


. . 80 


u 






1.0575 


. . 90 


a 






1.059 


. . 100 


u 



20 THE BLOOD 

Literature. — Schmaltz, Deutsch. Arch. f. klin. Med., vol. xlvii, p. 145; and 
Deutsch. med. Woch., 1891, No. 16. Stintzing u. Gumprecht, Deutsch. Arch. f. 
klin. Med., vol. liii, p. 265. Siegl, Prag. med. Woch., 1892, No. 20; and Wien. 
med. Woch., 1891, No. 33. Hammerschlag, ibid., 1890, p. 1018; and Zeit. f. 
klin. Med., 1892, vol. xxii, p. 475. Schmaltz, Deutsch. Arch. f. klin. Med., 1890, 
vol. xlvii, p. 145; and Deutsch. med. Woch., 1891, vol. xvii, p. 555. Appelbaum, 
Berl. klin. Woch., 1901, vol. xxxix, p. 7. 

Reaction. — The reaction of the blood during life, owing to the 
presence of disodium phosphate and sodium carbonate, is alkaline. 
The degree of alkalinity in healthy adults, while fasting, corresponds 
to about 300 to 325 mgrms. of sodium hydrate for 100 c.c. of blood 
(Lowy). Variations amounting to 75 mgrms. plus or minus are, 
however, not uncommon and in part due to unavoidable errors of 
technique (30 mgrms.). 

Generally the alkalinity of the blood is lower in women and children 
than in men, and is influenced by the process of digestion, exercise, 
etc. At the beginning of digestion, when hydrochloric acid is being 
freely secreted, the alkalinity of the blood increases; while later on 
it diminishes. Higher values are usually found during pregnancy 
than in the non-pregnant state. A decrease is observed following 
violent muscular exercise and also after the prolonged use of acids, 
while an increase is brought about by the ingestion of alkalies. An 
increase in the alkalinity of the blood occurs after a cold bath, and 
it is interesting to note that this is apparently associated with an 
increase in the bactericidal power of the blood. 

Under pathological conditions the alkalinity may be diminished or 
increased, as is shown in the table below. Unfortunately we are 
not able to account for these fluctuations in a satisfactory manner 
and the data are thus of little value. A marked decrease in diabetes 
may be viewed as of serious prognostic omen and as indicating acid 
intoxication. During diabetic coma the reaction owing to the pres- 
ence of large amounts of beta-oxybutyric acid may actually be acid. 
The supposition that in gout a diminished alkalinity exists in the 
intervals between attacks, and that this increases beyond the normal 
during the attack, has been proved unfounded. 

Orlowsky has recently expressed the opinion that the variations 
in the alkalinity of the blood which have been noted in various 
diseases and sometimes in one and the same disease, by various 
investigators working with the older methods, are referable to the 
varying tonicity of the blood and its varying richness in red corpuscles. 
Working with blood plasma Orlowsky found a marked diminution 
of the alkalinity in advanced uremia, in cancerous cachexia, and in 
severe cases of diabetes, while in other diseases normal values or 
at most but slight and exceptional variations were observed. 

The following table gives some of the results which have been 
obtained with Lowy's method: 



GENERAL CHARACTERISTICS OF THE BLOOD 21 

Carcinoma oesophagi 227-643 

Carcinoma ventriculi 256-635 

Ulcus ventriculi 302-460 

Anadeny of the stomach 354-360 

Alcoholic gastritis 343-379 

Chronic enteritis . 212-272 

Phthisis pulmonalis 450-468 

Bronchitis 239-343 

Neurasthenia 225-426 

Arteriosclerosis 208-344 

Chronic arthritis . . : 368-465 

Erysipelas , 498 

Typhoid fever 270-640 

Pneumonia 263-464 

Septicemia 443 

Leukemia . . . . . . . . - 368-835 

Pernicious anemia 429 

Diabetes mellitus . 362-457 

Chronic interstitial nephritis ........ 310-409 

Chronic parenchymatous nephritis 312-490 

Cirrhosis of the liver 272-345 

The alkalinity may be measured according to one of the following 
methods: 

Lowy's Method. — Five c.c. of blood, obtained from one of the 
superficial veins of the arm (preferably the median cephalic), are 
allowed to flow into a small flask provided with a long and partially 
graduated neck, and containing 45 c.c. of a 0.25 per cent, solution 
of ammonium oxalate. Coagulation is thus prevented and the blood 
made lake-colored — i. e., the hemoglobin is dissolved from the 
stroma of the red corpuscles. The mixture is then titrated with a 
2V normal solution of tartaric acid, using lacmoid paper, soaked in a 
concentrated solution of magnesium sulphate, as an indicator. 

As a normal solution of tartaric acid contains 75 grams to the 
liter, a -^ normal solution will contain 3 grains, and 1 c.c. of the -^ 
normal solution will corrrespond to 0.0016 gram of sodium hydrate. 1 

Supposing that 10 c.c. of the ^V normal solution were necessary 
to neutralize 5 c.c. of blood, the alkalinity of these 5 c.c. in terms 
of sodium hydrate would correspond to 0.016 gram, and the alka- 
linity of 100 c.c. of blood to 0.016X20 = 0.320 gram— i. e., to 320 
mgrms. 

Engel's Method. — This is essentially a modification of Lowy's 
method, and is well adapted for clinical purposes, as the amount of 
blood required for a single examination can readily be obtained by 
ordinary puncture. 

The blood is measured and rendered lake-colored in a specially 
constructed pipette (Fig. 1). To this end the blood is drawn to 
the 0.05 c.c. mark and diluted with neutral distilled water, so that 
the volume of the mixture reaches the 5 c.c. line. After slight agi- 

1 Regarding the standardization of normal solutions the reader is referred 
to special works on quantitative analysis. 



22 



THE BLOOD 



tation the solution is placed in a small beaker and titrated with a 
J- normal solution of tartaric acid, from a special burette which 
accompanies the pipette. This is so constructed that each cubic 
centimeter is divided into twenty parts. Before and after the 
addition of every drop of the titrating fluid the reaction of the mix- 
ture is tested by placing a drop upon lacmoid paper. The end 
reaction is reached when the yellow drop of the blood mixture shows 
a distinct red line along the margin. The result is expressed in terms 
of milligrams of sodium hydrate per 1 c.c. of blood. Normally about 
10 c.c. of the acid solution are employed. The tartaric 'acid solution 
contains 1 gram of Merck's crystals (crystallized reagent) to the liter, 
so that 1 c.c. corresponds to 0.533 mgrm. of sodium hydrate. 




Fig. 1. — Engel's alkalimeter. 

Supposing that 0.6 c.c. of the acid solution was required to neu- 
tralize the 0.05 c.c. of blood, then 12 c.c. would be necessary for 
1 c.c. of blood. As 1 c.c. of the acid solution represents 0.533 mgrm. 
of sodium hydrate, the alkalinity of 1 c.c. of blood would correspond 
to 12X0.533— i. e., to 6.396 mgrms. 

Dare's Method. — This method is based upon the fact that the 
characteristic spectrum of oxyhemoglobin disappears at the point 
of exact neutralization when the blood is titrated with a dilute solu- 
tion of tartaric acid. 

The examination is made with the aid of a special instrument, the 
hemo-alkalimeter, which is pictured in the accompanying illustration 
(Fig. 2). B is a glass stopper through which passes an automatic 
capillary blood pipette of 20 c.mm. capacity, the exposed end of 
which is groundto a tapering point, The stopper fits into the tube 



GENERAL CHARACTERISTICS OF THE BLOOD 



23 



A, which has a capacity of 3 c.c. and is graduated in cubic cen- 
timeters. The upper end of the tube is blown into a bulb with a 
minute aperture at C. A 2 c.c. dropping tube provided with a short 
piece of rubber tubing accompanies the instrument. 

To neutralize the blood a ^-q- normal solution of tartaric acid 
is used, which should contain an amount of alco- 
hol sufficient to prevent the growth of bacteria, but 
insufficient to precipitate the albumins of the blood. 
The reagent may be prepared by dissolving 0.075 
gram of tartaric acid (Merck's crystals; guaranteed 
reagent) in a small amount of distilled water, 
adding 20 c.c. of alcohol (93-94 per cent.), and 
diluting to 200 c.c. with water. 

For the spectroscopic examination a Browning 
instrument (Fig. 3) will suffice. 





Fig. 2. — Dare's hemo- 
alkalimeter. 




Fig. 3. — Browning's spectroscope. (Zeiss.) 



Method. — A drop of blood is obtained from the finger-tip or the 
lobe of the ear in the usual manner. The blood pipette is filled 
in situ by capillary attraction, holding the instrument horizontally 
to the drop of blood as it emerges from the wound. With an ordinary 
medicine dropper filled with distilled water the blood is washed 
into the bottom of the tube, connecting the dropper with the pipette 
by means of a short piece of rubber tubing. Blood and water should 
just reach the zero mark, and are intimately mixed by closing the 
aperture in the bulb with the finger and inverting the tube several 
times. The reagent pipette is then filled with the tartaric acid solu- 
tion and the rubber tubing slipped over the outer end of the blood 
pipette; by compressing the rubber bulb the acid solution is forced 
through the pipette into the test-tube, the aperture in the glass bulb 
being closed before the pressure is relaxed. Having done this the 
tube is inverted several times while still attached to the reagent 
pipette, taking care that this is held vertically so that the acid solu- 
tion does not get into the rubber bulb. The tube is clamped in front 



24 THE BLOOD 

of the spectroscope and examined for the two bands of oxyhemoglobin. 
(See Fig. 6.) So long as these are visible more of the acid is added, 
inverting the tube after each addition; as the bands become fainter 
one drop at a time is allowed to enter. At first this is rather tedious, 
but after several examinations have been made it will be found unneces- 
sary to apply the spectroscope so frequently to determine the point of 
neutralization, as the eye rapidly learns to recognize this by the 
characteristic change of color of the blood mixture. The observation 
is at an end when the oxyhemoglobin bands have just disap- 
peared. 

The examination is made with artificial light, keeping the distance 
from the light constant. 

Dare suggests that for sake of convenience the results be expressed 
in terms of the number of cubic centimeters of the tartaric acid 
solution instead of in mgrms. of sodium hydrate, as has been cus- 
tomary. The corresponding values are given in the table below, 
and have reference to 100 c.c. of blood. His normal values range 
between 266 and 292. 

Equivalent in terms 
C.c. of reagent : of mgrms. of NaOH 

per 100 c.c. of blood. 

2.6. ....../. 345.0 

2.4 319.0 

2.2 292.0 

.2.0 • 266.0 

1.8 239.0 

1.6 212.0 

1.4 176.0 

1.2 169.0 

1.0 133.0 

0.8 96.0 

0.6. 79.0 

0.4 . 53.0 

0.2 26.6 

Dare has ascertained with his method that there is a more or less 
constant relationship between the alkalinity of the blood and the 
color index, and he suggests that this may be the reason why the 
results obtained by different investigators differ so widely, as at 
different stages of the disease the color index may change. 

The method is quite convenient and merits the careful attention 
of all laboratory workers. 

Literature. — v. Jaksch, Zeit. f. klin. Med., 1887, vol. xiii, p. 350. A. Lowy. 
Arch. f. d. gesammte Physiol., 1894, vol. lviii, p. 462. Lowy u. Richter, Deutsch. 
med. Woch., 1895, vol. xx, p. 526. Peiper, Arch. f. path. Anat., 1889, vol. cxvi, 
p. 337. Rumpf, Centralbl. f. inn. Med., 1891, vol. xii, p. 447. Kraus, Arch, f . 
exp. Path. u. Pharmakol., vol. xxvi. Engel, Berlin, klin. Woch., 1898, p. 308. 
Brandenburg, Zeit. f. klin. Med., vol. xxxvi, p. 267. Orlowsky, Wratch, 1902, 
vol. xxii, pp. 1190 and 1222. A. Dare, Phila. Med. Jour., Jan. 17, 1903; and 
Johns Hopkins Hospital Bull., July, 1903. 



CHEMICAL EXAMINATION OF THE BLOOD 25 



CHEMICAL EXAMINATION OF THE BLOOD. 

Chemical Composition of the Blood. — A general idea of the 
chemical composition of the blood may be formed from the accom- 
panying table of C. Schmidt, calculated for 1000 parts: 

Man. Woman. 

Corpuscles ■ . 513.00 1 369.20 

Water 349.70 272.60 

Hemoglobin and globulins 159.60 120.10 

Mineral salts 3.70 3.55 

Plasma 486.90 603.80 

Water ............. 439.00 552.00 

Fibrin 3.90 1.91 

Albumins and extractives 39.90 44.79 

Mineral salts 4.14 5.07 

Blood plasma differs from blood serum in the presence of fibrinogen 
in the former and its absence in the latter. The substance is used up 
during coagulation, fibrin and a small amount of fibrinoglobuhn 
resulting. 

The albumins which are common to both plasma and serum are 
serum albumin and serum globulin. Of these the globulin is the 
larger fraction (3.84 as compared with 2.6 per cent., in horses' blood). 

From the following table it will be seen that a marked difference 
exists in the nature of the mineral ingredients between serum and 
the red corpuscles, the latter being relatively rich in potassium salts 
and phosphorus, and poor in sodium salts and chlorine. The figuies 
have reference to 1000 parts of blood 

Man. Woman. 

'"Red Red 

corpuscles. Serum. corpuscles. Serum. 

KoO 1.586 0.153 1.412 0.200. 

Na 2 0.241 1.661 0.648 1.916 

CaO . . 

MgO 

Fe 2 5 . . 

CI 0.898 1.722 0.362 1.440 

P.0 5 0.695 0.071 0.643 2.202 

It is noteworthy that the amount of sodium chloride in the serum, 
6 to 7 pro mille, remains fairly constant no matter whether large 
amounts are ingested or none at all is given. The term "isotonic" 
has been applied to a salt solution which is just strong enough to pre- 
vent the solvent action of the water upon the hemoglobin of the red 
corpuscles. In the case of the serum we meet with a condition of 
hyperisotonia — i. e., an amount of salt in excess of that actually 
required in order to prevent the destruction of the red corpuscles. 

1 This figure is too high; in man it varies between 420 and 470 for 1000 parts of 
blood. 



26 THE BLOOD 

Fat occurs in amounts varying from 1 to 7 pro mille in fasting 
animals, while following the ingestion of a meal rich in fats as much 
as 12.5 pro mille have been encountered. 

Soaps, cholesterin, and lecithin have likewise been found. 

Glucose is a normal constituent of the plasma, amounting to 
from 1 to 1.5 pro mille in man. While it is possible to increase this 
amount to a certain degree by the ingestion of large quantities of 
sugar, this appears in the urine, according to Claude Bernard, as 
soon as 3 pro mille have been exceeded. In addition to glucose, 
another reducing substance has been found in the blood, which differs 
from the former in not being fermentable. According to the re- 
searches of P. Mayer, 1 this is in all probability a glucuronic acid 
compound. Whether jecorin also occurs in the blood is doubtful. 

Among the extractives which have been found there may be men- 
tioned urea, uric acid, kreatin, carbamic acid, sarcolactic acid, gly- 
cogen, hippuric acid, and under pathological conditions xanthin, 
hypoxanthin, paraxanthin, adenin, guanin, leucin, tyrosin, lactic acid, 
cellulose, /3-oxybutyric acid, acetone, and biliary constituents. 

It has been pointed out that the color of the blood is referable to 
the presence of hemoglobin in the red corpuscles, and that it varies 
from a bright scarlet-red in the arteries to a dark bluish-red in the 
veins, the exact shade depending upon the amount of oxygen present 
in combination with hemoglobin as oxyhemoglobin. Upon chemical 
examination two other gases may be demonstrated under physiological 
conditions, viz., carbon dioxide and nitrogen. Of these, the latter 
appears to play no part in the body economy, and the amount present 
merely corresponds to that which would be absorbed by an equal 
volume of distilled water, viz., 1.8 vol. per cent., calculated at 0° C. 
and 760 Hgmm. pressure. 

The amount of oxygen and carbon dioxide, on the other hand, 
undergoes considerable variation, depending upon the particular 
bloodvessel from which the specimen is taken — i. e., whether this be 
an artery or a vein, and, furthermore, upon the velocity of the blood 
current, the temperature of the body, rest, exercise, etc. 

The relation existing between the amounts of these gases in arteries 
and veins may be seen from the following table : 

Arterial blood. Venous blood. 

Oxygen 21.6 per cent. 6.8 per cent. 

Carbon dioxide . . . 40.3 " 48.0 

Nitrogen 1.8 1.8 

Oxygen, as already pointed out, occurs principally in chemical 
combination with hemoglobin (oxyhemoglobin), only 0.26 per cent, 
being present in solution in the plasma. 

1 Zeit. f. physiol. Chem., vol. xxxii, p. 518. 



CHEMICAL EXAMINATION OF THE BLOOD 27 

Of the carbon dioxide which may be obtained from the blood, 
only one-tenth is held in solution. One-third is found in the red 
corpuscles, in the form of a loose compound with the alkalies of the 
corpuscles, and possibly also in combination with hemoglobin. The 
remaining portion is held in chemical combination by the alkalies of 
the plasma and albuminous bodies. 

Coagulation. — If blood is allowed to flow into a vessel and set 
aside, it will be observed at the expiration of a few minutes that the 
entire mass has become transformed into a semisolid, gelatinous 
material, which is spoken of as the blood clot or the placenta sanguinis. 
Still later it will be seen that a small amount of straw-colored fluid 
appears on top of the clot, which gradually increases in amount, while 
the clot itself undergoes shrinkage, until finally it floats, greatly 
diminished in size, in the surrounding fluid. The straw-colored 
fluid which has thus been obtained during the process of coagulation 
is spoken of as the blood serum. 

If a bit of the clot is examined microscopically, it will be seen to 
consist of a more or less dense network of fibers, the meshes of which 
are filled with blood corpuscles, which may be washed out, leaving 
the fibrous network, fibrin, behind. 

Chemically speaking, fibrin belongs to the class of the so-called 
coagulated albumins; it does not occur in the circulating blood, but 
is formed only during the process of coagulation. 

Under normal conditions blood clots in from two to six minutes 
after being shed, while in disease, notably in hemophilia, coagula- 
tion may be greatly retarded or does not occur at all, so that fatal 
hemorrhage may follow the infliction of trifling wounds. A ten- 
dency to hemorrhage is also observed in scurvy, purpura, in some 
infectious diseases, such as typhoid fever and yellow fever, in poison- 
ing with phosphorus, 1 etc. Sicard 2 has pointed out that in purpura 
primary coagulation occurs as with normal blood, but that sub-, 
sequent retraction of the clot and exudation of serum take place to 
only a very limited extent. Normal serum when added to fluids, 
such as hydrocele fluid, which are not spontaneously coagulable, 
in the proportion of 1 to 80, induces coagulation in from four to six 
hours. 

The serum of purpuric patients, on the other hand, is either 
entirely devoid of this property or possesses it to only a very slight 
degree. The addition of a trace of calcium chloride, however, causes 
such serum to behave very much like normal serum. Sicard hence 
suggests that in certain cases of purpura the fibrin ferment or its 
pro-enzyme is not present in sufficient quantity to cause more than a 
primary coagulation. 

1 Schmidt, Pfliiger's Archiv, vol. xi, pp. 291 arid 515. Bojanus, Inaug Diss., 
Dorpat, 1881. 

2 Compt.-rend. Soc. biolog., vol. li, p. 579. 



28 



THE BLOOD 



Wright's Coagulometer. — Wright's coagulometer may be con- 
veniently employed to determine the rapidity of coagulation. The 
instrument is shown in the accompanying illustration (Fig 4). The 
essential parts are a tin water can, a thermometer registered to about 
50° C, and a set of glass tubes measuring about 10 cm. in length with 
a lumen of 0.25 mm. and marked at 5 cm. When the instrument is 
to be used, the can is filled with water having a temperature about 
that of the body. The tubes are covered at their distal ends with 
little rubber caps and placed in their respective positions in the 
water bath, where they remain until they have acquired a similar 
temperature. 




Fig. 4. — Wright's coagulometer. 



They are then successively filled about one-half by aspiration 
from a drop of blood obtained from the finger or the lobe of the ear 
and replaced (properly numbered) tips down into the water in their 
proper positions. Careful note is kept of the exact time when they 
are filled. When a series of six or eight tubes has been filled, 
tube No. 1 is withdrawn from the water, and is held over a piece 
of white filter or blotting paper. The condition of the coagulation 
is then tested by blowing into the tube, the time of testing and the 
result being noted on the record. 



THE BLOOD PIGMENTS 29 

1. If the contents cannot be blown out, an entry is made on the 
record that coagulation is "complete." 

2. If the contents can be blown out, but if shreds of fibrin are 
found adhering to the inside of the tube or to the filter paper, coagula- 
tion is recorded as "incomplete/' 

3. If, lastly, the contents can be blown out cleanly, and if no trace 
of fibrin is seen on the filter paper, a note is made that coagulation 
has not yet begun. 

In the -first case the second tube is immediately taken in hand 
and is tested in the same manner as the first. If this is found clotted 
the next in series is tested, and so on, until a tube is found in which 
coagulation is still incomplete. 

In the second case, i. e., in the case where coagulation is found to 
be still incomplete, a slightly longer interval — reckoning from the 
time at which the second tube was filled in — is allowed to elapse 
before the tube next in series is tested. 

Lastly, if it is found that coagulation has not yet begun, an inter- 
val of one minute or more is allowed to elapse before testing the tube 
next in series. 

When the condition of the blood in a coagulation tube has been 
once tested, the tube in question must be put aside. Even if it has 
not all been blown out of the tube, its rate of coagulation will have 
been disturbed by the movement. 

Under normal conditions the coagulation time with these tubes 
will be found to vary between three and five minutes. The tempera- 
ture of the water in the can should be kept uniform during the exami- 
nation by adding hot water if necessary. 

The tubes are cleansed by removing the clots with a fine wire; they 
are then washed with water, with alcohol, and finally with ether. 



THE BLOOD PIGMENTS. 

Hemoglobin and Oxyhemoglobin.— Hemoglobin is a proteid 
which is composed of an* albuminous radicle, globin, and a non-albu- 
minous pigment radicle, hemochromogen. Upon the presence of the 
latter depends the readiness with which hemoglobin forms compounds 
with certain gases, such as oxygen, carbon monoxide, carbon dioxide, 
nitric oxide, and cyanogen. Hemochromogen in combination with 
oxygen is known as hematin. Oxyhemoglobin thus differs from 
hemoglobin merely in the fact that the pigment radicle is present in 
combination with oxygen. 

By itself hemoglobin is largely found in the blood of asphyxia. 
Under ordinary conditions it is principally present as oxyhemoglobin ; 
in arterial blood this preponderates, while in venous blood a mixture 
of both is found. 



30 



THE BLOOD 



On spectroscopic examination hemoglobin in suitable dilution 
shows a single band of absorption between D and E, extending slightly 
beyond D to the left (Fig. 5). 

Oxyhemoglobin shows two bands of absorption between D and 
E. One band, a, which is not so wide as the second, B, but darker 
and more sharply defined, borders on D; the second, which is wider 
but less sharply defined, lies at E (Fig 6). This spectrum can be 
readily transformed into that of hemoglobin by the addition of a re- 
ducing agent, such as ammoniacal solution of ferrous tartrate (Stokes' 
fluid), ammonium sulphide, or cuprous salts. 

Under normal conditions the amount of hemoglobin is fairly con- 
stant, but varies somewhat in different countries with the habits of 
the people, the character of the diet, etc. In Germany, as the result 
of 61 estimations, Leichtenstern found 14.16 per cent, by weight as 
the average in healthy men, and 13.10 per cent, in women. 



Red Orange Yellow 



Green 



Cyan-blue 



A a B C 

40 50 

lllllMillMllllllllIll 




Fig. 5. — Spectrum of reduced hemoglobin, (v. Jaksch.) 



Red Orange 



Yellow 



Green 



Cyan-blue 




Fig. 6. — Spectrum of oxyhemoglobin, (v. Jaksch.) 

Clinically we express the amount of hemoglobin by relative figures 
as compared with the average normal percentage by weight; on this 
basis the scale of the various hemoglobinometers is constructed. On 
these instruments the figure 100 represents the average normal value; 
this, however, varies somewhat with the various forms of hemo- 
globinometers according to the average percentage by weight which 
has been taken as a standard in establishing the 100 mark. With 
the Gowers instrument Strauss and Rohnstein obtained figures varying 
between 85 and 125 as normal values; this would furnish an average 
of 105. Schaumann and v. Willebrandt give 88 as the average 
normal. With the v. Fleischl instrument I rarely find higher values 
than 90 per cent, in inhabitants of large cities, but with the Dare 
apparatus the average results more nearly approach the 100 mark. 

In children the average values are somewhat lower than in the 



THE BLOOD PIGMENTS 31 

adult. Stierlin gives 79.7 per cent, for boys and 82.1 for girls. 
Borchmann's values are even lower, viz., 55 and 80; Gundobin gives 
70 and 95. 

The ingestion of large amounts of water does not cause a dilution 
of the blood and hence a diminution of the amount of hemoglobin; 
but relatively higher values are found upon the withdrawal of liquids, 
owing to a concentration of the blood as a whole. Fat persons show 
smaller values than correspond to their age. 

Pathological Variations. — Abnormally high values, hyperchromemia, 
viz., 120 to 150 per cent., occur in cases of chronic enterogenous 
cyanosis, and may also be observed in congenital heart disease. 
The hyperchromemia in these cases is associated with polyglobulism. 

A pathological decrease is spoken of as oligochromemia, and is 
observed in all forms of anemia from whatever cause. 

The lowest values are found in chlorosis, in which the oligochrome- 
mia far exceeds the oligocythemia, viz., the diminution in the number 
of the red cells. In an analysis of 94 cases I found an average of 
42.5 per cent.; the lowest value was 17.5 (Fleischl). There are 
instances on record in which the reading was still lower. 

Very low figures are seen in splenic anemia, and it is rare, excepting 
in chlorosis, to find such a low grade of chromemia associated with 
a blood count which is normal or may indeed be above normal. The 
average of 13 estimations given by Osier was 47 per cent. 

In pernicious anemia the oligocythemia exceeds the oligochro- 
memia. The loss of hemoglobin is, however, also quite marked 
and may be as great as in the most extreme cases of chlorosis. In 
the series of 23 cases collected by Strauss and Rohnstein the average 
value was 25 per cent. (Gowers) ; in 9 cases it was lower than 20 per 
cent. A. Meyer reports a bothriocephalus case with only 10 per cent. 

In the early stages of leukemia the loss of hemoglobin is often not 
especially marked; later the anemia may become quite intense, but 
the oligochromemia is not necessarily of high grade even in well- 
developed cases. Ehrlich cites cases in which the Gowers instrument 
gave readings of from 60 to 70 per cent. On the other hand, there are 
cases in which the oligochromemia is an early feature of the disease, 
and in one instance of this kind I obtained a reading of only 27 per 
cent. Cases of this order have been described as leukanemia. The 
blood picture is essentially a composite of leukemia and pernicious 
anemia. 

While in the course of typhoid fever the amount of hemoglobin is 
always reduced (Osier), and usually to a greater extent than the 
number of the red corpuscles, the most severe grades of anemia 
may be encountered during convalescence, when the amount of 
hemoglobin may fall to 20 per cent. 

In the early stages of carcinoma of the stomach the cachexia is not 
well pronounced. Schiile states that in his analysis of 198 cases it 



32 THE BLOOD 

occurred in only 30 per cent. Later the loss of hemoglobin is quite 
marked; the values may indeed approach those seen in chlorosis and 
pernicious anemia. 

An intense grade of anemia is seen in generalized septicemia, and, as 
Ewing remarks, no form of the acute disease appears to act more 
violently than does puerperal or uterine sepsis. A diminution in 
the amount of hemoglobin to 20 per cent, is here not uncommon. In 
the chronic cases also a high grade of oligochromemia is a constant 
feature. In a case of lumbar abscess of six months' duration I found 
21 per cent, of hemoglobin, with 1,025,000 red cells. The hemo- 
globin in all these cases diminishes more rapidly than the number of 
the red cells. 

In pulmonary tuberculosis a diminution in the amount of hemo- 
globin is seen essentially in the third stage of the disease (40 to 45 per 
cent.), while previously fairly normal values are obtained (90 to 95 
per cent.). It is to be noted, however, that a certain grade of anemia 
(69 per cent.) is quite commonly observed, even in the first stage, in 
those cases in which the disease has been of very gradual onset, viz., 
in patients who often have suffered from tuberculous affections (scrof- 
ula) since childhood. In the third stage the anemia is well marked 
(40 to 50 per cent.). 

A notable diminution in the amount of hemoglobin is observed in 
chronic nephritis, chronic enteritis, in chronic lead and mercurial 
poisoning, in syphilis, etc. 

In syphilis the anemia develops at a time when the entire organism 
has been thoroughly infected. The lowest hemoglobin values are 
reached just before or coincidently with the appearance of the rash. 
In the secondary stage the degree of oligochromemia, cceteris "paribus, 
may be regarded as a fair index of the severity of the infc ction. In 
untreated cases the hemoglobin remains low for several days or even 
for weeks. A gradual rise then occurs which is associated with 
beginning involution of the exanthem. In uncomplicated cases 
normal values may subsequently be reached even without treatment; 
a fall again occurs with relapses. Similar changes are observed in the 
tertiary stage. Especially interesting are the observations of Justus 
on the blood changes which occur in the course of mercurial treat- 
ment; Justus ascertained that a rapid and material diminution of 
the hemoglobin (10 to 20 per cent.) occurs when a large (medicinal) 
amount of mercury is introduced at one time into the body of the 
infected individual. This decrease is only observed in the blood of 
patients with florid syphilis; it is specific and does not occur in healthy 
nor in otherwise diseased individuals. The reaction is demonstrable 
in every form of syphilitic infection (secondary, tertiary, and heredi- 
tary) as soon as the more distant lymph glands begin to swell. It 
disappears, or is at least no longer demonstrable, with beginning 
involution of the symptoms. 



THE BLOOD PIGMENTS 33 

The practical value of this syphilitic blood test has not yet been 
definitely established. While some observers have expressed them- 
selves against its value, it must be recognized that in discussing his 
adversaries' criticisms Justus seems to have maintained the upper 
hand. 

During anesthesia by ether the amount of hemoglobin is always 
absolutely reduced. In some instances there is an apparent increase, 
but this is never proportionate to the rise in the number of the red 
cells which is simultaneously observed (Da Costa, Kalteyer). Owing 
to the hemocytolysis which thus undoubtedly takes place a very low 
percentage of hemoglobin should be regarded as a counterindication 
to general anesthesia. A lower value than 50 per cent, is now re- 
garded by many as a dangerous figure. 

For the estimation of hemoglobin see p. 147. 

Literature. — Strauss u. Rohnstein, Die Blutzusammensetzung b. d. verschied- 
enen Anaemien, Hirschwald, Berlin, 1901. Appelbaum, Berl. klin. Woch., 1901, 
vol. xxxix, p. 7. Quincke, " Zur Pathologie d. Brutes," Deutsch. Arch. f. klin. 
Med., vols, xxv and xxvii. Leichtenstern, Unters. iiber d. Hemoglobingehalt d. 
Blutesim gesunden u. kranken Zustande, Leipzig, 1878. W. Osier, "On Splenic 
Anemia," Am. Jour. Med. Sci., 1902, vol. cxxiv, p. 763. Justus, Virchow's Archiv, 
vol. cxl, p. 1; and Deutsch. Arch. f. klin. Med., 1902, vol. lxxv, p. 1. 

Hemoglobinemia. — The term hemoglobinemia has been applied 
to a condition in which the hemoglobin is dissolved out from the red 
corpuscles, and, appearing in the plasma as such, leads at first to a 
very decided choluria and in extreme cases to hemoglobinuria. 

Various poisons, such as potassium chlorate, carbolic acid, pyro- 
gallic acid, naphtol, arsenic, antimony, hydrochloric acid, sulphuric 
acid, antifebrin, antipyrin, phenacetin, sulphonal, tincture of iodine, 
when given hypodermically, or even internally in sufficiently large 
doses, will call forth a hemoglobinemia which is followed by hemo- 
globinuria. 

Fresh morels also contain a poison which is capable of producing 
an intense hemoglobinuria, and which may be extracted with hot 
water. 

In acute and chronic infectious diseases of a severe type, such as 
scarlatina, typhoid fever, intermittent fever, icterus gravis, syphilis, as 
also in diseases depending upon a hemorrhagic diathesis, such as 
variola hemorrhagica, scurvy, as also following insolation, extensive 
burns, and frostbite, hemoglobinemia, leading to hemoglobinuria, is 
not infrequently observed. The same has been noted in splenic 
anemia and in Raynaud's disease. In syphilis a moderate grade of 
hemoglobinemia can be demonstrated by spectroscopic examination 
of the serum within two or three minutes following an intravenous 
injection of mercuric chloride in medicinal doses. (See also Justus' 
test.) 

An epidemic hemoglobinuria of the newly born and a paroxysmal 
3 



34 



THE BLOOD 



or intermittent hemoglobinuria, both of unknown origin, have like- 
wise been described. 

Hemoglobinemia also follows the infusion of blood of animals of 
one species into the circulation of animals of a different species. 

In some cases, and particularly in those following poisoning with 
chlorates, etc., the hemoglobinemia ultimately leads to a well-pro- 
nounced methemoglobinemia (see below). 

A hemoglobinemia, aside from the urinary examination, may be 
readily recognized by a spectroscopic examination of the serum, when 
the two bands of absorption of oxyhemoglobin will be observed. 

A very simple method which may be employed for the same purpose 
is the following: One-half to 1 c.c. of blood is collected in a small 
glass tube, drawn out and sealed at one end. This amount can be 
readily obtained by puncturing the ear and milking out the blood, 
which is transferred to the tube by means of a little pipette. After 
the blood has clotted, the clot is separated from the walls by means 
of a wire or a glass rod and the corpuscles packed down by centri- 
fugation. With normal serum the supernatant fluid presents a 
straw-yellow color, while in hemoglobinemia it is colored a more pr 
less intense red. 

If the supernatant fluid is withdrawn, diluted with a little water, 
and heated to 70 to 80° C, the coagulum in the presence of hemoglobin 
will present a brownish color. 

Literature. — Ponfick, Verhandl. d. Cong. f. inn. Med., 1883, vol. ii, p. 205. 
Stadelmann, Arch. f. exp. Path. u. Pharmakol., 1882, vol. xv, p. 337, and 1884, 
vol. xvi, pp. 118 and 221. Afanassiew, Zeit. f. klin. Med., 1883, vol. vi, p. 281. 
v. Jaksch, Verhandl. d. Cong. f. inn. Med., 1891, vol. x, p. 353. 

Carbon Monoxide Hemoglobin.— In cases of coal-gas poisoning 
the blood, both of arteries and veins, presents a bright cherry-red 
color, owing to the presence of carbon monoxide hemoglobin. 

Such blood, when properly diluted, like oxyhemoglobin, shows two 
bands of absorption between D and E (Fig. 7), which are nearer the 



Bed Orange 



Yellow 



Green 



Cyan-blue 



a B 

i0 



Eb 



I 1 I I 1 I, 



i0 70 

n 



90 100 110 

i i il ii nliiiiji i i il i i iil iiii 



Fig. 7.— Spectrum of carbon monoxide hemoglobin, (v. Jaksch.) 



violet end of the spectrum, however, and may readily be distinguished 
from those referable to oxyhemoglobin by the addition of a reducing 
agent. This will not affect the spectrum of carbon monoxide hemo- 
globin, while that of oxyhemoglobin is transformed into the spectrum 
of reduced hemoglobin. 



THE BLOOD PIGMENTS 35 

For medico-legal purposes a number of additional tests have been 
devised, among which that suggested by Hoppe-Seyler is one of the 
simplest and at the same time most reliable. The blood is treated 
with double its volume of a solution of sodium hydrate (sp. gr. 1.3). 
Normal blood is thus changed into a dirty-brownish mass, which 
exhibits a trace of green when spread upon a porcelain plate, while 
carbon monoxide blood yields a beautiful red under the same con- 
ditions. 

Nitric Oxide Hemoglobin. — The blood in cases of poisoning 
with nitric oxide, owing to the presence of nitric oxide hemoglobin, 
yields a spectrum which is similar to that of carbon monoxide hemo- 
globin; the bands, however, are less sharply defined and paler than 
those of the latter, and, like these, do not disappear on the addition 
of a reducing substance. 

Sulphohemoglobin (Methemoglobin Sulphide). — In cases of 
poisoning with hydrogen sulphide no definite changes can be dis- 
covered in the blood upon spectroscopic examination, although 
Hoppe-Seyler has shown that hemoglobin may enter into com- 
bination with this gas. It is stated, however, that in such cases the 
blood becomes dark and of a dull-greenish tint, and that the distinc- 
tion between arterial and venous blood is lost. 

A remarkable instance of sulphohemoglobinemia has been de- 
scribed by v. d. Berg, 1 in a case of autotoxic enterogenous cyanosis. 
In this case an organism producing hydrogen sulphide was isolated 
from the stools. When grown in a solution of normal oxyhemoglobin 
sulphohemoglobin resulted. 

Carbon Dioxide Hemoglobin. — With carbon dioxide, as men- 
tioned above, hemoglobin is also thought to enter into combination, 
the spectrum being similar to that of reduced hemoglobin. The 
latter, in fact, is formed artificially when carbon dioxide is passed 
through a solution of oxyhemoglobin. If this process is carried 
farther, the hemoglobin is decomposed and globin is thrown down; 
an absorption band is then obtained which is similar to that result- 
ing when hemoglobin is decomposed with acids (see below), and is 
no doubt referable to the presence of free hemochromogen. 

Of the blood changes occurring in cases of poisoning with hydro- 
cyanic acid and acetylene but little is known, and the reader is referred 
to works on toxicology for their consideration. 

Hematin. — If oxyhemoglobin in aqueous solution is heated to a 
temperature of from 60° to 70° C, it is decomposed into globin and 
hematin. The same result is reached by treating the aqueous solu- 
tion with acids, alkalies, or the salts of various heavy metals. 

Hematin is an amorphous, blackish-brown, or bluish-black sub- 
stance which is frequently encountered in old transudates, in the 

1 Arch. f. klin. Med., 1905, vol. lxxxiii, p. 86. 



36 



THE BLOOD 



stools after hemorrhages, and after meals consisting largely of red 
meats. It is said to occur in the urine in cases of poisoning with 
arsenic, and in the blood of animals poisoned with nitrobenzol its 
presence can likewise be demonstrated with the spectroscope. 



Red Orange 

A 



Yellow 



Green 



Cyan-blue 



t a 


B C 


D 




Eb 


F 






40 50 


60 


70 


80 


90 100 


ll 


Mil 


llllllllllllllllll 


iimIiim 


iimIimiIi, 


^J 


II 1 ll 1 llll II ll 1 III 1 1 1 1 


1 1 1 1 1 1 



















Fig. 8. — Spectrum of hematin in alkaline solution, (v. Jaksch.) 

In acid solution it shows a well-defined spectral band between 
C and D (Fig. 10). Between D and F a second band is seen, which 
is much wider but less sharply defined than the first, and may be 
resolved into two bands by dilution, one between b and F, near F, 
and another between D and E, near E; a faint fourth band may 
also be seen between D and E, near D. As a rule only the two 
bands between D and F are visible. 



Bed Orange 
A 



Yellow 



Green 



djan-blve 



a B 

40 



LmI.m.ImmI. 



Innlnnl, 



Eb 

80 

mIim 



i.n.i 



100 110 

llill lli l 



Fig. 



-Spectrum of reduced hematin. (v. Jaksch.) 



In alkaline solutions it shows but one broad band, the greater 
portion of which lies between C and D, extending slightly beyond 
D (Fig. 7). 

If an alkaline solution of hematin is treated with a reducing sub- 
stance, reduced hematin (hemochromogen) results, which gives 
rise to two absorption bands between D and E (Fig. 9). 

Hemin. — Hematin readily combines with one molecule of hydro- 
chloric acid to form hemin. This substance crystallizes in light- 
brown or dark-brown rhombic plates or columns, which are quite 
characteristic (Plate I). They bear the name of their discoverer, 
Teichmann. The size of these crystals varies with the manner in 
which they are produced, the largest specimens being met with when 
the glacial acetic acid (see below) is allowed to evaporate as slowly 
as possible. Specimens measuring from 15/>« to 18 J^ in length may 
then be seen. Smaller crystals will be present at the same time, 
occurring either singly or in the form of stars, rosettes, and crosses. 

As these crystals may be obtained from mere traces of blood, their 



PLATE I. 




Hemin Crystals. 



THE BLOOD PIGMENTS 



37 



formation must be regarded as conclusive evidence in medico-legal 
examinations. Lewiji and Rosenstein have pointed out, however, 
that under certain conditions a negative result may be reached, even 
if the coloring matter is derived from the blood. This is the case 
especially when the hemoglobin has been transformed into hemo- 
chromogen or hematoporphyrin, or when substances have been 
mixed with the blood which are either capable of altering its general 
composition or which, through their mere presence, interfere with the 
reaction. Such substances are certain salts of iron (rust), lead, mer- 
cury, and silver; further, lime, animal charcoal, and sand, when inti- 
mately mixed with the blood. In medico-legal cases a spectroscopic 
examination should hence be made whenever the hemin reaction is 
not obtained. 

Method. — A small drop of normal salt solution is carefully 
evaporated on a slide, when a few particles of the suspected material, 
powdered or teased as finely as possible, are placed on the delicate 
layer of crystallized salt. Glacial acetic acid is now added drop by 
drop and the specimen carefully heated (three-quarters to one minute) 
until bubbles begin to form. While evaporation is being continued 
glacial acetic acid is further added until a light-brown tint appears. 
As soon as this point is reached, the last traces of the acid are allowed 
to evaporate, the specimen being held at a greater distance from the 
flame. A drop of glycerin is then added and the preparation covered 
with a cover-glass. The examination is made with a one-fifth or 
a one-sixth objective. Attention is especially directed to brownish 
streaks or specks, which, in the presence of blood, can usually be 
made out with the naked eye. 

Methemoglobin. — Methemoglobin is a pigment closely related to 
oxyhemoglobin, and is frequently encountered in hemorrhagic transu- 
dates, cystic fluids, and in the urine in cases of hematuria and hemo- 
globinuria. In the circulating blood methemoglobin is found after 



Red Orange Yellow 



A a B C 



Green 



Cyan-biu*. 



Eb 



40 50 60 70 






.IhmImmLmLmI 



90 100 110 

■ ImmImmIm 



Fig. 10.— Spectrum of methemoglobin in acid and neutral solutions, (v. Jaksch.) 



the ingestion of large quantities of potassium chlorate, notably in 
children, as also after the inhalation of nitrate of amyl, the use of 
kairin, thallin, hydrochinon, pyrocatechin, iodine, bromine, turpen- 
tine, ether, perosmic acid, permanganate of potassium, and antifebrin 
(see Hemoglobinemia). Most remarkable is the occurrence of met- 
hemoglobinemia in cases of so-called autotoxic enterogenous cyanosis, 



38 



THE BLOOD 



as reported by Stokvis and v. d. Berg. In one case the latter found 
sulphohemoglobin in the place of methemoglobin. 

The spectrum of an aqueous or slightly acidified solution of met- 
hemoglobin (Fig. 10) closely resembles that of an acid solution of 
hematin, but differs from this in the ease with which it is transformed 
into that of hemoglobin when an alkali and a reducing substance are 
added. The spectrum of hematin under the same conditions is trans- 
formed into that of an alkaline solution of hemochromogen. In 
alkaline solutions, on the other hand, two bands of absorption are 
observed, which are similar to those of oxyhemoglobin, but differ 
from these in the fact that the band nearer E, b, is more pronounced 
than the one at D, a. A third, but very faint, band may further be 
observed between C and D, near D. 

Hematoidin. — Small amorphous particles of an orange or ruby- 
red color, or crystals belonging to the rhombic system, occurring 
either singly or in groups, are frequently met with in the sputum, 
the urine, and the feces, as well as in old extravasations of blood. 
They were discovered by Virchow, who applied the term hematoidin 
to this particular pigment, the hemic origin of which is undoubted. 
It is supposedly identical with bilirubin. 

Hematoporphyrin. — Hematoporphyrin is likewise a derivative of 
hematin, and, according to Nencki and Sieber, isomeric with biliru- 
bin. In dilute solution with sodium carbonate it shows four bands 



Red Orange Yellow 



Green 



Cyan-blue 




Fig. 11. — Spectrum of hematoporphyrin in alkaline solution. 

of absorption, one between C and D; a second one, broader than the 
first, about D, especially marked between D and E; a third one, not 
so broad and less sharply defined, between D and E, and a fourth 
one, broad and dark, between b and F (Fig. 11). 

The clinical significance of this body, which also appears in the 
urine, as well as the causes which give rise to its formation, are 
unknown (see Hematoporphyrinuria) . It has been found postmortem 
in the blood, in a case of sulphonal poisoning, by Taylor and Sailer. 1 



1 A. E. Taylor and J. Sailer, Contrib. from the William Pepper Laboratory, 
Phila., 1900, p. 120. 



THE PROTEINS OF THE BLOOD 39 



THE PROTEINS OF THE BLOOD. 

In considering the proteins of the blood from a clinical point of 
view, it is necessary to distinguish between an increase and a dimi- 
nution in their normal amount, constituting the conditions of hyper- 
albuminosis and hypalbuminosis, respectively. As may be expected, 
the former is met with whenever water is more rapidly withdrawn 
from the system than it can be supplied, and is hence observed in 
cases of cholera, acute diarrhea, following the use of purgatives, etc. 
This increase in the amount of proteins is only a relative increase, 
however. The occurrence of an absolute increase has not been 
satisfactorily demonstrated. An absolute hypalbuminosis, on the 
other hand, is observed following a direct loss of proteins from 
the blood, as in hemorrhage, dysentery, albuminuria of high degree, 
the formation of large collections of pus, etc. This is generally 
associated with a relative increase in the amount of water— i. e., a 
hydremia — which is particularly noticeable after hemorrhages, and 
referable to a diminished secretion and excretion of water, as well 
as to a direct absorption from the tissues. Hypalbuminosis has also 
been observed in pernicious anemia, and is dependent partly upon 
a diminution in the amount of the albumins of the serum and partly 
upon a decrease in the weight of the corpuscular solids. The amount 
of serum-albumin is about normal, while the globulins are much 
diminished. 1 

The term hyperinosis has been applied to a condition in which the 
amount of fibrin (normally 0.349 to 0.425 per cent.) is increased. 
This is said to occur in various inflammatory diseases, such as pneu- 
monia, pleurisy, scarlatina, acute articular rheumatism, and ery- 
sipelas, while a diminished amount of fibrin, hypinosis, or normal 
values are seen in malaria, nephritis, pyemia, pernicious anemia, 
typhoid fever, and leukemia (both lymphoid and myeloid). 

In order to determine the amount of fibrin, 30 to 40 c.c. of blood, 
obtained by means of cupping glasses or venesection, are placed in a 
previously weighed beaker, fitted with an India-rubber cap, through 
the centre of which passes a piece of whalebone, firmly fixed. The 
blood is defibrinated by beating with the whalebone, when the beaker 
with its contents is weighed, the difference indicating the weight of the 
blood. The beaker is then filled with water and the mixture again 
beaten. The fibrin is allowed to settle and after being washed 
with normal salt solution collected on a filter of known weight. 
It is further washed with normal salt solution until free from color- 
ing matter, then boiled in alcohol to dissolve out fat, cholesterin, 
and lecithin, dried at 110° to 120° C, and on cooling weighed over 
sulphuric acid. 

1 Erben, Zeit. f. klin. Med., 1900, vol. xl, p. 266. 



40 THE BLOOD 

Fairly satisfactory results may also be obtained by simply making 
wet mounts (which see), ringing with vaselin and setting aside for 
several hours, when they are examined microscopically. In cases of 
pneumonia and acute articular rheumatism marked fibrin formation 
will be observed, starting from clumps of blood platelets. 

The presence of albumoses and peptone bodies in the blood of 
leukemic (myeloid) patients has been repeatedly observed after the 
blood has stood for some time, or after the death of the patient 
(v. Jaksch. 1 Matthes, 2 Erben, 3 Schumm 4 ). Their formation is due 
to the liberation of a proteolytic ferment, derived from the polynuclear 
neutrophiles. Schumm also found leucin and tyrosin. In normal 
human blood Schumm found no albumoses after death. In intersti- 
tial nephritis a fair amount could be demonstrated. 

Albumoses have also been found in a case of abscess of the brain, 
associated with albumosuria. Freund 5 claims that they are met with 
in sarcoma, while they are absent in carcinoma (not confirmed). 

Following the injection of nuclein and spermin albumosemia 
appears to occur quite constantly both during the stage of hypo- 
as well as hyperleukocytosis. After injections of pilocarpin albumo- 
suria is observed only in association with hyperleukocytosis. 

In order to test for albumoses, the coagulable albumins should first 
be removed, when a positive biuret reaction in the filtrate will indicate 
their presence (see also Salkowski's test). 

Carbohydrates . Sugar. — Sugar, as indicated above, is a normal con- 
stituent of the blood, its quantity varying between 1 and 1.5 pro mille. 
Under pathological conditions this amount may be exceeded and 
notably so in diabetes, in which Hoppe-Seyler found as much as 9 pro 
mille in a given case. 

In addition to sugar, a non-fermentable reducing substance has 
been encountered in the blood, which, according to Mayer, appears 
to be a compound glucuronate. 6 The presence of jecorin in the 
blood still remains to be proved. 

Large quantities of a reducing substance, the greater portion of 
which consisted of sugar, have been met with by Trinkler in carci- 
noma; it was observed at the same time that carcinoma of inter- 
nal organs was associated with far greater amounts of sugar than 
cancerous disease of the skin and the mucous membranes. It is 
also interesting to note in this connection that an increase in the 
degree of the cachexia was not accompanied by an increase in the 
percentage of sugar. 

1 Zeit. f. physiol. Chem., vol. xvi, p. 243. 

2 Berlin, klin. Woch., 1894, Nos. 23 and 24. 

3 Zeit. f. Heilk., 1903, vol. xxiv. 

4 Hofmeister's Beit., vol. v, p. 442. 

5 Freund u, Obermayer, Zeit. f. physiol. Chem., vol. xv, p. 310. 

6 Ibid., vol. xxix, p. 59. 



THE PROTEINS OF THE BLOOD 41 

The results reached by Trinkler 1 apparently also bear out the 
correctness of the conclusions formed by Freund, who claimed that 
a differential diagnosis between carcinoma and sarcoma, in which 
latter condition no increase in the amount of sugar was noted, can 
always be effected upon the basis of an examination of the blood 
in this direction. Further examinations in this direction are lacking. 

In the following table the percentages found in the different dis- 
eases investigated are given, from which it is apparent that, next to 
carcinoma, the largest quantities of sugar are met with in the infec- 
tious diseases and the lowest figures in diseases of the kidneys : 

Average. Minimum. Maximum. 
Per cent. Per cent. Per cent. 

Carcinoma ....... 0.1819 0.1023 0.3030 

Typhoid fever 0.0950 0.0875 0.1022 

Pneumonia 0.0943 0.0813 0.1092 

Dysentery ....... 0.0838 0.0796 0.0915 

Heart disease 0.0737 0.0664 0.0897 

Peritonitis 0.0701 0.0450 0.0917 

Tuberculosis 0.0653 0.0450 0.0817 

Syphilis ......... 0.0553 0.0449 0.0748 

Nephritis and uremia .... 0.0489 0.0321 0.0559 

In order to demonstrate sugar in the blood, 15 to 30 grams, ob-' 
tained by venesection or cupping glasses, are placed in an evapo- 
rating dish and treated with an equal weight of finely powdered 
sodium sulphate and a few drops of acetic acid. The mixture is 
brought to the boiling point and passed through a muslin filter as 
soon as the coagulum has become black and spongy, water having 
previously been added to the original volume. The filtrate is passed 
through Swedish paper. In the final filtrate the sugar is then esti- 
mated as described elsewhere (see Urine). 

Cavazzani has drawn attention to another method of freeing the 
blood from proteids, which is said to be entirely satisfactory. To 
this end; 20 to 30 c.c. of blood are added to 200 c.c. of distilled 
water in a porcelain dish and treated with 5 or 6 drops of a solution 
consisting of 10 parts of acetic acid (sp. gr. 1.040) and 1 part of 
lactic acid. The mixture is boiled for eight to ten minutes, filtered, 
and the coagulum washed repeatedly with hot water and finally pressed 
out in a piece of muslin. The resulting filtrates, which are prac- 
tically colorless, are then concentrated to a small volume, and any 
traces of albumin, which may still separate out, filtered off. If an 
excess of the acid solution has been added, it may happen that the 
mixture does not clear up on boiling. It is then only necessary to 
add a few crystals of sodium carbonate, when coagulation will occur 
at once. On the other hand, it may at times be necessary to add a 
few more drops of the acetic acid solution. 

1 Centralbl. f. d. med. Wiss., 1890, p. 498. Freund u. Obermayer, loc. cit. 



42 THE BLOOD 

Williamson's Diabetic Blood Test. — This test is of much interest, 
and may possibly serve to differentiate the ordinary forms of diabetes 
from that in which the blood sugar is not increased. It is based 
uponl the f observation that a warm alkaline solution of methylene 
blue is decolorized by grape sugar. A positive result may at times 
be obtained when the sugar has temporarily disappeared from the 
urine. 1 

Method. — Twenty cbmm. of blood, obtained from the finger or 
the ear, are carefully measured off with the aid of the capillary pipette, 
which accompanies Gower's hemocytometer, and mixed in a test- 
tube of small caliber with 40 cbmm. of distilled water. To this 
mixture 1 c.c. of an aqueous solution of methylene blue to 6000) 
and 40 cbmm. of a 6 per cent, aqueous solution of potassium hydrate 
are added. A control tube is similarly charged with non-diabetic 
blood. The two specimens are placed in boiling water and allowed 
to remain for three to four minutes, without shaking. At the end 
of this time it will be seen that the diabetic blood has decolorized 
the methylene-blue solution, which has turned a dirty yellowish 
green or yellow, while the non-diabetic specimen has retained its 
original color. 

The quantity of blood used should not exceed the amount indi- 
cated, as a decolorization of the methylene blue also results with 
non-diabetic blood if large amounts, such as 60 cbmm., are em- 
ployed. 

The reaction is supposedly due to an increase of glucose in the 
blood, and was obtained in all of forty-three cases of diabetes which 
were examined. It is said to be obtainable for a considerable time 
after death. Adler 2 found the reaction in all of nine cases of dia- 
betes, while in one hundred and twenty-one non-diabetic cases nega- 
tive results were reached. Very curiously, it was absent in non- 
diabetic glycosurias. Adler believes the reaction to be referable to 
a diminished alkalinity of the blood. 

Glycogen. — There appears to be no doubt that glycogen normally 
occurs in the blood of various animals. Huppert 3 succeeded in 
demonstrating its presence in all animals examined, the amount vary- 
ing between 0.114 and 1.560 grams for 100 parts of blood (see 
Iodophilia). 

Cellulose.— Cellulose has been found in the blood of tuberculous 
patients. 

Urea. — Urea occurs normally in the blood in traces — 0.016 to 
0.020 per cent. Larger amounts are encountered whenever, as 
in nephritis, various diseases of the urinary organs, cholera Asiatica, 
cholera infantum, eclampsia, etc., its elimination is impeded, or when- 

1 R. T. Williamson, Centralbl. f. inn. Med., vol. xviii, No. 33. 

2 Zeit. f. Heilk., 1900, vol. xxi, No. 11. 

3 Zeit. f. physiol. Chem., 1893, vol. xviii, p. 144. 



THE PROTEINS OF THE BLOOD 43 

ever, as in fever, owing to increased albuminous decomposition, 
urea is formed in abnormally large quantities. 

It is interesting to note that a smaller amount of urea is found in 
fatal cases of eclampsia than in those ending in recovery, which has 
been explained by the assumption that in this condition the functional 
activity, not only of the kidneys, but also of the liver, is lost. 

The methods which are available for the detection of urea in the 
blood are still too complicated for clinical purposes, and the value 
of the information derived so small as hardly to warrant the labor 
involved. Hoppe-Seyler's method should be employed whenever an 
examination in this direction is deemed advisable. 1 

Uremia. — Formerly, it was thought that the complex of symp- 
toms generally spoken of as uremia was referable to the retention 
in the blood of urea or ammonium carbonate. This view has since 
been disproved, although it must be admitted that in uremia an 
increased amount of urea is frequently noted. Other views, accord- 
ing to which uremia is referable to an accumulation of potassium 
salts, of extractives, and especially of kreatinin, or of ptomams in 
the blood, must be regarded as being sub judice. There is no reason, 
however, to ascribe the uremic condition to the retention in the blood 
of one particular constituent of the urine, and it is not improbable 
that a retention of all may be responsible for the symptoms observed. 

Literature. — Feltz and Ritter, De Puremie exper., Paris, 1881. Astaschewsky, 
St. Petersburg med. Woch., 1881, No. 27. Bouchard, Lecons sur l'autointoxica- 
tion, Paris, 1887. Rovighi, Rivista clinica, 1886. 

Ammonia. — Normal venous blood, according to the researches 
of Winterberg, contains about 1 mgrm. of ammonia for each 100 c.c. 
In febrile conditions variable results are obtained, but it appears 
certain that a definite relation between the height of the fever and the 
amount of ammonia does not exist. In chronic hepatic diseases, 
and notably in cirrhosis, it is not increased. Acute yellow atrophy 
also is not necessarily associated with an increase. Very significant 
is the observation that in uremia following extirpation of the kid- 
neys no increase is observed. An ammoniemia in the sense of v. 
Jaksch can hence scarcely be said to exist. 

Literature. — Nencki, Pawlow, and Zaleski, Arch. f. exp. Path. u. Pharmakol., 
1896, vol. xxxvii, p. 26. Winterberg, Wien. klin. Woch., 1897, p. 330. 

Uric Acid and the Xanthin Bases. Uric Acid.— Formerly, the 
presence of appreciable amounts of uric acid in the blood was regarded 
as pathognomonic of gout. But we now know that a lithemic con- 

1 See Hoppe-Seyler, Handbuch der physiologisch und pathologisch-chemischen 
Analyse. 



44 THE BLOOD 

dition may occur also in other diseases. Traces of uric acid are indeed 
encountered under normal conditions. 

A definite lithemia has been observed in a variety of disorders, 
such as pneumonia, acute and chronic nephritis, leukemia, conditions 
associated with an insufficient aeration of the blood, as in the various 
diseases of the heart, in pleurisy with exudation, emphysema when 
accompanied by cyanosis, the severer forms of anemia, etc. v. Jaksch 
claims to have found uric acid in the blood in 88.88 per cent, of his 
cases of nephritis. Fever in itself does not appear to lead to an 
increased production of uric acid, as negative results were obtained 
in nine cases of typhoid fever out of eleven, in five cases of acute 
articular rheumatism out of six, etc. 

The assumption that acute attacks of gout are referable to increased 
alkalinity of the blood, and a consequent increase in the amount 
of circulating uric acid, has been disproved. 

In order to estimate the amount of uric acid in the blood, the fol- 
lowing method may be employed: 100 c.c. of blood, obtained by means 
of venesection or of cupping glasses, are at once diluted with three or 
four times their volume of water and heated on a water bath. As 
soon as coagulation sets in, a few drops of a 0.3 to 0.5 per cent, 
solution of acetic acid are added until a feebly acid reaction is obtained. 
After having been kept upon the boiling water bath for from fifteen 
to twenty minutes longer, until the albumin has separated out and 
settled in brownish flakes, the mixture is filtered while hot, and 
the precipitate washed repeatedly with hot water. Filtrate and 
washings, which usually present a slightly yellow or brownish color, 
are again brought to the boiling point after the addition of 0.3 to 
0.5 per cent, of acetic acid, decanted, filtered, and after the addition 
of a small amount of disodic phosphate further treated according 
to Folin's method (see Urine). 

Literature. — Picard, Virchow's Archiv. vol. ii, p. 189. Garrod, Med.-Chir. 
Trans., 1854, p. 49. Salomon, Zeit. f. physiol. Chem., vol. ii, p. 65; and Charite 
Annalen, 1880, vol. v, p. 137. Klemperer, Deutsch. med. Woch., 1895, No. 40. 
Weintraud, ibid., V. B. p. 185. 

Xanthin Bases. — Xanthin bases do not occur in normal blood or 
are present only in exceedingly small amounts. Under pathological 
conditions they may be encountered in recognizable quantities, 
so in leukemia, typhoid fever, lymphatic tuberculosis, emphysema, 
phthisis pulmonalis, pleurisy, and chronic nephritis. 

To demonstrate the xanthin bases in the blood the albumins are 
first removed as just described (see Uric Acid) and the filtrate then 
examined according to Salkowski's method (see Urine). 

Literature. — A. Kossel, Zeit. f. physiol. Chem., 1882, vol. vii, p. 22. Scherer, 
Verhandl. d. physik. med. Ges. z. Wiirzburg, 1852, vol. ii, p. 325. 



THE PROTEINS OF THE BLOOD 45 

Fat and Fatty Acids. — Engelhardt has pointed out that the amount 
of fat which is contained in normal human blood may be subject to 
considerable variations, and gives 0.194 per cent, as the average. 
The lowest figure which he obtained was 0.101 and the highest 
0.273 per cent. These figures differ very materially from those of 
older observers, who have found from 0.73 to 1.4 per cent., but it 
is quite likely that Engelhardt's method is responsible for these 
differences, and is probably more reliable (see below). Unfortunately 
only a few analyses of pathological material have been made with 
this method, and these have reference only to the blood of cachectic 




Fig. 12. — Pronounced lipemia. Specimen treated with osmic acid. Lower half shows extra- 
cellular fat globules, upper half having been cleared by oil of turpentine. (Gumprecht.) 

individuals. An increase in the amount of fat has here not been 
demonstrated, the results varying between 0.112 and 0.284 per cent., 
with 0.174 as an average. The cachexias in question were of tuber- 
culous and carcinomatous origin. With the older methods an increase 
in the amount of fat, aside from that observed after the ingestion 
of large amounts of fatty food, has been met with in cases of obesity, 
chronic alcoholism, in phosphorus poisoning, in injuries affecting 
the long bones and the spinal cord, in various hepatic diseases, chronic 
nephritis, tuberculosis, malaria, cholera, during starvation, pregnancy, 
in nursing infants, etc. The greatest increase, however, is observed 
in certain cases of severe diabetes, in which amounts varying between 
1.276 and 18.12 per cent, have been encountered, and in which the 
fat may be visible with the naked eye (see below). In such cases 
fat emboli may be found postmortem, plugging the vessels of various 



46 THE BLOOD 

organs, and notably the brain, the lungs, and the kidneys. This 
increase in the amount of fat constitutes the condition spoken of as 
lipemia. 

The term lipacidemia has been applied to the occurrence of fatty 
acids in the blood. This has been noted in various febrile diseases, 
leukemia, and especially in grave cases of diabetes, where beta- 
oxybutyric acid may be found in large amounts, and is no doubt 
directly concerned in the production of coma. 

To demonstrate the presence of fat in the blood, it is best to pre- 
pare cover-glass specimens, and to mount these in a drop of a 5 per 
cent, solution of osmic acid. The fat droplets are thus colored 
black, and appear about as large as the finest fat granules which are 
found in milk or butter. They may also be stained with Sudan III, 
or Biebrich scarlet, and are thus colored red. In every case the 
necessary instruments and glasses should be carefully cleansed with 
ether, so as to avoid the accidental introduction of fat. 

As a quantitative estimation of the fat is not always possible, 
Landy recommends the following simple procedure to demonstrate 
the presence of an excess of fat: A small drop of blood is received 
upon a cover-glass, which is then adjusted over the depression of a 
cupped slide and ringed with vaselin. On standing, the serum 
separates out concentrically or excentrically from the small blood 
clot, and normally or in the presence of no excess of fat appears 
perfectly clear. If, however, much fat is present, it becomes cloudy 
after several minutes or hours, and then appears bluish- white, gray- 
ish-white, or even milky-white. To ascertain positively that the 
turbidity is due to fat, a microscopic examination of the hanging 
drop is made within a few hours following the preparation of the 
specimen, so as to exclude fibrin as the possible cause of such tur- 
bidity. 

Quantitative Estimation. — The apparatus which is best used is 
a modification of that of Nerking, as suggested by Engelhardt. 1 
As seen from Fig. 13, it consists of the ether flask A, which is placed 
on a permanent water bath, such as that of Miinke. a represents 
the escape tube for the ether vapor; at b there is a closure by means 
of mercury, the upper escape tube c dipping into the mercury over 
the mouth of b. B is the cooler for the ether vapor; C, the water 
condenser. The cooled ether falls through the cooler into d. This 
ends below with a funnel-shaped mouth, close to the bottom of the 
extraction flask E, with five apertures, and has a small open side 
tube, /, which counteracts any negative pressure that may occur 
above the liquid in the extraction flask. The fluid to be extracted 
extends to within 1 to 2 cm. from the aperture of the off-flow tube i. 
When the ether layer extends to the level with k the tube i acts as a 

1 The apparatus may be procured from Arno Haak, Jena. Price, 12 marks. 



THE PROTEINS OF THE BLOOD 



47 



siphon and draws off the fatty ether into A again by way of the tube 
I, which is likewise provided with a mercury stop. 

The blood, about 10 c.c., is received in a graduate and weighed. 
It is washed into the extraction flask with about ten times its volume 
of 2 per cent, hydrochloric acid and boiled for three hours (with 
inverted condenser). On cooling, the material is extracted in the appa- 
ratus described for about forty-eight hours. At the expiration of 
this time the fatty ether in A is poured into a separating funnel 
together with the ethereal washings, 
which are used to remove all the 
material from the flask, the idea 
being to get rid of any water or bits 
of the bloody material that may by 
chance have been siphoned into A. 
The ether is then evaporated in an 
open glass dish. The residue is dis- 
solved in absolute ether and filtered 
through a double folded filter (so as 
to absorb any traces of water remain- 
ing) into a beaker, when the ether is 
allowed to evaporate. The residue is 
placed in a drying oven at 40° C. for 
one hour, and after remaining in the 
vacuum over sulphuric acid for twelve 
hours it is weighed. 

With this method lecithins, choles- 
terins, and fatty acids are obtained 
conjointly with the fat, which Engel- 
hardt does not regard as objectionable, 
as they are present only in traces and 
may be regarded as physiologically 
equivalent to neutral fat. 

Estimation of Fatty Acids. — This is 
carried out along the same lines as des- 
cribed in the Urine (Lipaciduria), after 
removal of the coagulable albumins. At 
least 20 to 30 c.c. should be available. 

Cholesterin. — Traces of cholesterin 
are normally met with in the blood. 

observed in diabetes (0.478 per cent.) in association with marked 
lipemia. 1 

Hale White 2 reports a case in which microscopic examination showed 
a granular precipitate, which did not stain with osmic acid. Chemi- 




Fig. 13.^Fat-extraction apparatus. 



Larger amounts have been 



1 Virchow's Archiv, vol. clxxii, Heft 1 and 2. 

2 Lancet, October 10, 1903. 



48 THE BLOOD 

cal examination led to the conclusion that the substance was an ester 
of cholesterin with one or more of the higher fatty acids. 

Literature. — M. Bonninger, " On the Methods for the Estimation of Fat in the 
Blood, and the Amount of Fat in Human Blood," Zeit. f. klin. Med., vol. xlii, 
parts i and ii. T. B. Futcher, " Lipemia in Diabetes Mellitus," Jour. Am. Med. 
Assoc, 1899, p. 1006. S. Watjoff , " Ueber d. Fettgehalt d. Blutes b. Nierenkrank- 
heiten," Deutsch. med. Woch., 1897, p. 559. v. Jaksch, "Lipacidsemie," Zeit. f . 
klin. Med., vol. xi. W. Ebstein, " Beitrag z. Lehre v. d. Lipemie u. d. Fettembolie," 
etc., Virchow's Archiv, 1899, vol. civ, p. 571. M. Engelhardt, Deutsch. Arch. f. 
klin. Med., 1901, vol lxx, p. 182. Zandy, ibid., vol. lxx, p. 301. 

Lactic Acid. — There appears to be some doubt whether or not 
lactic acid normally occurs in the blood of man during life. In the 
blood of dogs, Gaglio, could always demonstrate the presence of the 
acid during the process of digestion, after feeding with meat. The 
amount varied between 0.3 and 0.5 pro mille. During starvation 
smaller amounts were found, but it never disappeared altogether. 
In one instance Gaglio obtained 0.17 pro mille after fasting for 
forty-eight hours. Similar results were obtained by Irisawa, who 
noted that the amount of lactic acid in the blood stood in direct 
relation to the degree of anemia which was produced. 

In the human being Irisawa found lactic acid fairly constantly 
after death, the amount, determined as zinc lactate, varying between 
0.233 and 6.575 pro mille. These extensive variations he was unable 
to explain by the character of the disease causing the fatal termina- 
tion, and it is possible that the cause lies in the fact that in some 
cases the blood was obtained shortly after death, while in others 
many hours had elapsed, as Irisawa himself suggests, v 

The following method may be employed: 100 to 300 c.c. of blood 
are extracted with three times its volume of alcohol, filtered, and 
the filtrate evaporated to a syrupy consistence. This is then made 
strongly alkaline with barium hydrate and shaken with large quan- 
tities of ether, in order to remove the fats which are present. The 
residue is acidified with phosphoric acid and again shaken with ether 
for twenty minutes at a time, until the process has been repeated 
five or six times, the lactic acid passing over into the ether. The 
ether is distilled off from the extract, the residue taken up with 
water, and the solution carefully evaporated in order to drive off any 
ether still remaining, as well as the fatty acids. Carbonate of zinc 
is now added and the solution heated to 100° C. and filtered. The 
filtrate is evaporated on a water bath until crystallization begins, 
when it is allowed to cool and treated with a few drops of absolute 
alcohol, in order to effect a complete separation of the lactate of zinc. 
The solution is allowed to stand exposed to the air until a constant 
weight is obtained. 

Literature. — G. Gaglio, "Die Milchsaure d. Blutes," Du Bois Archiv., 1886, 
p. 400. T. Irisawa, "Ueber d. Milchsaure im Blut und Harn," Zeit. f. physiol. 
Chem., 1892, vol. xvii, p. 349. 



THE PROTEINS OF THE BLOOD 49 

Homogentisinic Acid. — Homogentisinic acid has been demon- 
strated in the blood serum of an alkaptonuria, by Abderhalden and 
Falta. 1 

Biliary Constituents and Urobilin.— Bile pigment does not occur 
in the blood under normal conditions, but may be demonstrated 
whenever it is present in the urine (obstructive jaundice, hepatic 
cirrhosis, acute yellow atrophy, phosphorus poisoning, etc.). It 
appears, moreover, that bilirubin is present in the blood in nearly 
every case where urobilin is found in the urine. In pernicious 
anemia bilirubinemia is thus quite constantly associated with uro- 
bilinuria. At the same time urobilin can usually be demonstrated 
in the blood. 

In chlorosis bile pigment does not occur in the blood. 

The demonstration of bilirubinemia constitutes the most delicate 
test for the entrance of bile into the blood and lymph; it is a much 
more delicate indication than the occurrence of bilirubinuria. 

Bilirubin can be demonstrated in the blood most readily in the fol- 
lowing manner: A short piece of glass tubing is drawn out so as to 
form a tapering lower end, which is then sealed. By means of a pi- 
pette 10 to 15 drops of blood, obtained by free puncture of the finger 
or ear, are transferred to the first tube and the serum separated from 
the corpuscles by centrif ligation. The coagulum which forms is sepa- 
rated from the walls and packed down into the lower portion of the 
tube. The supernatant fluid is normally clear or but faintly turbid, 
and of a straw color: in the presence of bilirubin it is colored a bright 
yellow, which on exposure to the air gradually turns to a greenish 
tint. 

For more exact information the method of Syllaba may be used : 10 
to 15 c.c. of blood are placed in a cool place for sedimentation. The 
serum which separates out is removed with a pipette and 5 c.c. 
diluted with double the amount of water and coagulated by boiling 
after the addition of a pinch of sodium sulphate and acidifying 
with dilute acetic acid. Any bilirubin that may be present is carried 
down in the coagulating albumin while urobilin remains in solution. 
The fluid is then filtered and the filtrate tested by boiling to make 
sure that the coagulation is complete. 

If no urobilin is present the filtrate is clear, colorless and spectro- 
scopically free from absorption ; if, ho were;*, urobilin is present in the 
serum, as is usually the case in pernicious anemia, then the filtrate 
presents a reddish color and shows a narrow band of absorption between 
b and F. The collected precipitate in the absence of bilirubin (in 
normal serum and the serum of chlorosis) is white, but in the presence 
of bilirubin (in the serum of pernicious anemia) of a slight yellowish 
color. The precipitate is washed with hot water, boiled with acidu- 

1 Zeit. f. phys. Chem., vol xxxix, p. 143. 



50 THE BLOOD 

lated alcohol (sulphuric acid) and the mixture filtered. In the 
presence of bilirubin the alcohol is colored a fine green and the 
coagulum presents the same color; in the absence of bilirubin the 
alcohol remains colorless. 

In order to test for biliary acids, the blood is first treated with 
alcohol, in order to remove the proteids. The biliary acids which 
are present in the filtrate are next transformed into their lead salts 
by means of lead acetate and ammonia and thus precipitated. 
After washing with water the precipitate is boiled with alcohol and 
filtered. The lead salts are decomposed by means of sodium carbo- 
nate, the solution is again filtered, the filtrate evaporated to dryness, 
and the residue extracted with absolute alcohol. The alcohol is dis- 
tilled off, when the biliary salts of sodium will crystallize out or 
remain behind as an amorphous mass, which may be tested directly 
according to Pettenkoffer's method. To this end, some of the residue 
is dissolved in water and treated with two-thirds of its volume of 
concentrated sulphuric acid, care being taken that the temperature 
does not rise beyond 60° C. To this mixture a few drops of a 20 
per cent, solution of cane sugar are added, when in the presence of 
biliary acids a beautiful violet color is obtained, which is referable 
to the action of furfurol, formed from the cane sugar and the acid, 
upon the biliary acids. 

Acetone. Acetone has been found in the blood in considerable 

amounts under various pathological conditions, and especially in 
diabetes and fevers. 

In order to demonstrate its presence, Dennige's test may be 
employed: 3 c.c. of blood are treated with about 30 c.c. of Dennige's 
reagent and allowed to stand until the dark-brown precipitate has 
settled to the bottom. The supernatant fluid is filtered off and 
treated with a little more of the reagent, so as to ensure complete 
precipitation. It is then acidified with sulphuric acid and heated as 
described. The formation of a white precipitate, which is soluble 
in an excess of hydrochloric acid, is referable to acetone or diacetic 
acid. (See Urine.) 

Literature. — v. Jaksch, Acetonurie u. Diaceturie, Berlin, 1885. Reale, 
Schmidt's Jahrbuch., 1892, p. 106 (Extract). 

Choiin. — Cholin has been demonstrated by Moth and Halli- 
burton in the blood in diseases of the nervous system which are 
associated with a destruction of nerve tissue; notably in general 
paresis, tabes, combined sclerosis, disseminated sclerosis, alcoholic 
polyneuritis, beriberi, and following the division of both sciatic 
nerves in cats, 

Method. — Five c.c. of blood are treated with from six to eight 
times that amount of absolute alcohol and filtered. The filtrate is 
dried at 40° C, and the dry residue extracted three times with absolute 



MICROSCOPIC EXAMINATION OF THE BLOOD 51 

alcohol, filtered, and the solution evaporated. The alcoholic solution 
of the residue is precipitated with a 10 per cent, alcoholic solution of 
platinum chloride and the precipitate decanted from the absolute 
alcohol. The precipitate is finally dissolved in 15 per cent, alcohol, 
the solution filtered and evaporated in a watch crystal at 40° C. 
With a low power the octahedral crystals of cholin-platinochloride 
can then be seen. 

Normal human blood (in the amount mentioned) rarely gives rise to 
such crystals, so that the result is practically negative. Sine qua 
non for the success of the method is that the alcohol is absolute; 
99 per cent, will not suffice. [See also Donath's method (Cerebro- 
spinal fluid)]. 



MICROSCOPIC EXAMINATION OF THE BLOOD. 

The Red Corpuscles. Variations in Size and Form. — The normal 
red blood corpuscles are greenish-yellow, circular bodies, which in 
postembryonic life are non-nucleated. While it has been generally 
accepted that the red cells are biconcave several writers have recently 
insisted that they are bell-shaped (YVeidenreich, F. T. Lewis). They 
are possibly composed of fluid contents within a membrane of some 
fatty substance. Their diameter varies between 6 and 9 p., with an 
average of 7.5 y-. The presence of larger or smaller cells is abnormal. 
Smaller cells are termed microcytes and measure from 3.5 to 6 fi; larger 
cells are known as macrocytes or megalocytes, and usually have a diam- 
eter of from 9.5 to 12 u.\ still larger specimens are spoken of as giant 
corpuscles (Hayem) ;* they may attain a diameter of 16 p.. The terms 
microcytosis or microcythemia and macrocytosis or macrocythemia are 
used to designate a predominance of the corresponding variety. 

As regards the origin of the macrocytes, there is evidence to show 
that they may result from the common normocytes in the circulating 
blood through imbibition of water, so that their occurrence from 
this point of view could be regarded as a degenerative phenom- 
enon. But, on the other hand, the presence of macrocytes may be 
interpreted as evidence of a regenerative process, bearing in mind 
that in the bone-marrow the size of the erythroblasts is larger than 
that of the common normocytes; the macrocytes would thus repre- 
sent young normocytes which have prematurely found their way 
into the circulation. The microcytes probably result from the normo- 
cytes in the circulating blood through loss of water; whether their 
presence may at any time be regarded as the expression of a regen- 
erative process seems questionable. Not infrequently microcytes 
are formed artificially during the preparation of the specimen. 

1 Le Sang, Paris, 1891. 



52 THE BLOOD 

Microcytosis is, on the whole, of comparatively little clinical 
interest, and may be observed in any severe anemia. Macrocytosis 
is more important. To a certain extent it is seen in severe forms 
of anemia of whatever origin, but it is noteworthy that the presence 
of macrocytes in large numbers is essentially observed in pernicious 
anemia. During the active period of the disease the macrocytes 
may here represent 70 per cent, of all red cells (Lazarus). The 
condition, however, is not constant. 

Going hand in hand with pathological variations in the size of the 
red corpuscles — anisocytosis — there are variations in form which may 
affect not only the microcytes and macrocytes, but also the corpuscles 
of normal size. Cells may thus be seen which resemble a flask, a 
kidney, a biscuit, a boat, a balloon, a dumb-bell, or an anvil, while 
others are altogether irregular in appearance. 






'J* 



Fig. 14. — Poikilocytosis. 

Especially interesting is the fact that such abnormally formed cells, 
which are generally spoken of as poikilocytes (Fig. 14), may manifest 
a certain degree of motility, so that they have at times been mistaken 
for microparasites. This is seen especially in marked cases of per- 
nicious anemia, and is most noticeable in the smaller forms. In 
pernicious anemia poikilocytosis is most pronounced, and at one 
time it was thought that the condition was characteristic of the disease. 
It has been shown, however, that it occurs in other anemias as well, 
though its occurrence is probably always evidence of a specially 
severe form. In chlorosis it is usually only seen in the most severe 
cases, and particularly in those manifesting a tendency to throm- 
bosis and embolism. 

In this connection a special deviation from the normal form of the 
red corpuscles also requires consideration, viz., the prevalence of 



PLATE II 




L.S. 



The Elements of Normal Blood- 



ed red cells in rouleaux; b, crenated red cells; c, finely granular (neutrophilic) leukocytes; d, 
coarsely granular (eosinophilic) leukocytes; e, small, and /, large mononuclear leukocytes; 
g, plaques. 



MICROSCOPIC EXAMINATION OF THE BLOOD 53 

oval cells. These are notably observed in pernicious anemia and 
seem to be of distinct diagnostic importance. They are found not 
only during the active periods of the disease, but frequently also in 
the interval between exacerbations. 

Poikilocytosis is a degenerative phenomenon, and it is essential 
not to confound true poikilocytes with certain abnormal forms, 
which may be seen in any normal preparation and which are the result 
of mechanical injury, mutual compression, etc., and can readily be 
distinguished with practice. 

In wet preparations red cells will be seen near the margin of the 
drop where evaporation is actively going on, which present little 
knobs or spicules on their surface and along the periphery. Such 
cells are spoken of as crenated cells. The phenomenon in itself is 
normal, but it is noteworthy that crenation may at times be observed 
in the centre of a carefully prepared specimen after a few seconds 
already, while as a rule from fifteen to thirty minutes elapse before 
the process begins to attack cells in this location. The significance 
of this early crenation is not known. This is also true of delayed 
money-roll formation, which is observed in various hepatic diseases, 
in pneumonia, nephritis, etc., whereas normally the red corpuscles 
tend to agglutinate in this form immediately unless special pains 
have been taken to secure the separation of the individual cells. 
(See Plate II.) 

Variations in the Color of the Red Corpuscles. — The degree of color- 
ing of the red corpuscles depends upon the amount of hemoglobin. 
The centres of the cells in well-mounted specimens are always paler 
than the periphery, and any deficiency in the amount of coloring 
matter is here at once apparent. With a moderate grade of anemia 
the cell as a whole looks paler, and the pale central area is increased 
in size. With a further increase in the loss of coloring matter the 
central area is absolutely colorless and encroaches upon the peripheral 
colored zone more and more until finally the so-called pessary forms 
result, in which only a narrow rim of hemoglobin remains. These 
changes can be made out in wet preparations, but are especially 
well seen in stained specimens. The central pale area is, however, 
visible only in well-preserved cells and not in flattened out cells, 
which are stained uniformly throughout and which may also be seen 
in any specimen. 

The color of the normal red cells in wet specimens is a pale greenish- 
yellow. In malaria curiously discolored corpuscles are seen, which 
present a bronzed appearance; their presence should always excite 
suspicion. The meaning of the discoloration is not known, but in all 
probability it is evidence of a degenerative process. 

The Color Index. — The term color index is used to designate the 
relative amount of hemoglobin which is contained in each corpuscle. 
It is determined by dividing the percentage of blood coloring matter 



54 THE BLOOD 

by the percentage of red cells as compared with the recognized 
normal, viz., 5,000,000. 

Example. — The percentage of hemoglobin is 50, the red count 
per cbmm. is 2,000,000, viz., 40 per cent, of the recognized normal, 
5,000,000. The color index is then 50 divided by 40— i. e., 1.25. 

Under normal conditions the color index is about 1, but may vary 
from 0.95 to 1.17; it is slightly higher in men than in women. In 
the secondary anemias, in which the decrease in the amount of 
hemoglobin is proportionate to the diminution of the red corpuscles, 
the color index is approximately normal. But in the majority of 
cases the diminution of the hemoglobin somewhat exceeds that of 
the red cells, so that lower values are commonly met with. In 
pernicious anemia, on the other hand, where the corpuscular decrease 
usually exceeds the diminution of the hemoglobin, a high color index 
is the rule. There may be periods in the course of the disease, how- 
ever, in which a normal index and even subnormal values are found. 
In the chronic cases lower figures are more commonly obtained than 
in the acute cases. In the series of 22 cases collected by Strauss and 
Rohnstein the value of the color index varied between 0.5 and 1.95. 
In 8 cases of the series variations from 1.13 to 1.95 were observed, 
and in 6 lower values than 1 were noted, viz., 0.5 to 0.9. Cases in 
which the color index falls as low as 0.5 are rare in pernicious anemia. 
In the one instance in the series in which this was found, the hemo- 
globin was only 10 per cent., while the red cells numbered 1,048,000; 
there was a high grade of poikilocytosis and all transitions between 
the smallest microcytes and the largest types of macrocytes. 

In well-established hookworm anemia in contradistinction to the 
cryptogenetic type of pernicious anemia the color index is low 
(Ashford). 

In the secondary anemia of carcinomatosis the color index rarely 
exceeds 1. In Strauss and Rohnstein's series 1 of 35 cases the highest 
value was 1.1 (in one case only); in the rest it varied between 0.53 
and 0.96. 

In chlorosis, in which the degree of oligochromemia exceeds the 
corpuscular loss the color index is markedly lowered; in especially 
severe cases it may fall to 0.3 and even lower. But it is not ad- 
missible to make the diagnosis of chlorosis on this basis only, as it is 
fairly common to meet with a markedly lowered color index in some 
secondary anemias also, and especially in the form which is referable 
to carcinoma, as has just been mentioned. In splenic anemia like- 
wise the degree of oligochromemia may far exceed the degree of 
oligocythemia. 

Variations in Number. — The number of red corpuscles in the 
blood of healthy adults is fairly constant. In man 5,000,000 may 

1 Die Blutzusammensetzung b. d. verschiedenen Anemien. Berlin, 1891. 



MICROSCOPIC EXAMINATION OF THE BLOOD 55 

be considered a fair average, and in women 4,500,000. Higher 
values are not uncommon, bat rarely exceed 6,000,000 in perfectly 
normal individuals. 

The largest number is found on the first day after birth, average 
6,985,428. It diminishes until the third day. Following a temporary 
rise it drops farther and becomes fairly constant between the sixth 
and the tenth day. 1 

In 20 healthy infants Karnizki 2 obtained the following values: 

Age. 

2-4 months . 5,239,725 

4-8 " 5,703,000 to 5,843,000 

8-12 " . . . ."■ . . . . . 5,531,000 10 5,590,521 

Then the number increases, especially after the sixth year, and 
remains on an average higher during childhood than in babyhood. 

A somewhat higher average is found among people living at a 
considerable elevation above the sea level, and it is interesting to 
note that an increase in the number occurs whenever a change in 
the habitation is made from a lower to a higher level. This increase 
is frequently quite marked, as is apparent from the following table, 
which is taken from Ehrlich: 3 

Altitude Increase of 

561 meters . . . ' . . . 800,000 

700 " 1,000,000 

1800 " 2,000,000 

4392 " 3,000,000 

A corresponding diminution occurs when a change is made from a 
higher to a lower level. 

In this connection Gaule's 4 observations are of interest. On 
the occasion of a balloon ascension to a height of from 4200 to 4700 
meters he counted 7,040,000, 8,800,000, and 7,480,000, respectively, 
in the three participants of the journey. The hemoglobin was at 
the same time diminished, and he accordingly concluded that the 
increase during the ascent was due to an increased production of red 
cells; the probable nature of this conclusion was strengthened by the 
fact that numerous normoblasts were found in the blood, many under- 
going division. Jolly and Bensaude 5 and others on similar expeditions 
were unable, however, to demonstrate the presence of nucleated red 
cells or to note the occurrence of an increased number of red cells. 
According to Weinzirl, 8 the increased counts due to high altitude are 

1 Scipiades, Arch. f. Gyn., vol. ixx, p. 630. 

2 Arch. f. Kinderheilk., 1903, vol. xxxvi. 

3 "Die Anamie," Nothnagel's specielle Path. u. Therap., vol. viii, part. i. 

4 Compt.-rend., vol. cxxxiii, p. 903. 

5 Compt.-rend. Soc biol., vol. liii, p. 1084. Saint Martin, Soc de biol., July 
23, 1904 

6 Amer. Jour. Med. Sci., 1903, vol. cxxvi, p 299. 



56 THE BLOOD 

temporary and in part at least referable to cold. He showed that in 
rabbits a certain increase in the number of red cells occurs when they 
are removed from warm to cold quarters, and that their subsequent 
removal to a higher altitude does not lead to a further increase. 

Clinically we distinguish between relative "polycythemia in which 
the condition is due to a diminution in the quantity of plasma, and 
true polycythemia in which there is an actual increase in the number 
of the red corpuscles. Relative polycythemia is much the more 
common. In some instances it is due to loss of liquid by sweating, 
diarrhea, or increased diuresis. In another group of cases there is loss 
of liquid by secretion or transudation, as in obstruction of the pylorus 
with dilatation of the stomach, and in the constant loss of liquid 
from the blood in recurring ascites. In some of these cases the 
polycythemia is of high grade, and may persist for years. In advanced 
cases of nephritis, phthisis, malignant disease, etc., there is also a 
certain grade of relative polycythemia, due to loss of water from the 
body at large. The polycythemia which is noted in poisoning by 
phosphorus and carbon monoxide (in one case of coal-gas poisoning 
a count of 11,200,000 is reported), various coal-tar products, 
during and immediately after the administration of ether, following 
cold baths and severe muscular exercise, also belongs to this order and 
is no doubt referable to vasomotor disturbances. Of similar origin 
probably is the polycythemia which is noted in disease of the adrenal 
glands, where counts of from 6,000,000 to 7,000,000 have been 
repeatedly noted; and the same is probably true of diabetes, in 
which polcythemia may be observed both while fasting and while 
much fluid is being ingested. 

True polycythemia is met with in diseases in which there is diffi- 
culty in proper aeration of the blood, as in heart disease, 1 and in a 
peculiar type of chronic cyanosis which has been described by 
Osier 2 as a new clinical entity, the so-called autotoxic enterogenous 
cyanosis. In acquired heart disease with continued inadequacy of 
the circulation of slight degree a moderate grade of polycythemia 
is very common; in the congenital form the figures often reach 
8,000,000 to 9,000,000. The highest values are seen in Osier's 
disease, however. In the first nine cases which have been reported the 
highest count was 12,000,000; in eight it was above 9,000,000, and in 
the ninth it was 8,250,000. The usual range of hemoglobin at the same 
time was from 120 to 150; the specific gravity varied between 1.067 
and 1.083, and the leukocyte count between 4000 and 20.000; as a 
rule it was below 10,000. Vaquez 3 notes that whereas in congenital 
heart disease and the coincident polyglobulism the diameter of the 

1 Stengel, Proc. Path. Soc. Phila., 1899. Oertel, Deutsch. Arch., vol. i, p. 293. 

2 " Chronic Cyanosis with Polycythemia," Amer. Jour. Med. Sci., 1903, vol 
cxxvi, p. 187. 

3 Soc. biol., 7 Mai. 1892 



MICROSCOPIC EXAMINATION OF THE BLOOD 57 

red cells is increased from 7.5 to 8.5, this is not observed in the idio- 
pathic form of cyanosis. 

While there can thus be no doubt that a true polycythemia does 
occur, it has been conclusively demonstrated that such a condition 
does not exist in what is generally termed plethora, and that the 
various symptoms of plethora formerly attributed to a geneial increase 
in the amount of blood are referable to vasomotor disturbances. 

Oligocythemia, viz., a diminished number of red cells, is much 
more common than polycythemia. It may be temporary or perma- 
nent, and is seen in all forms of anemia of whatever origin. It is 
most marked in pernicious anemia. The exact figure will here, of 
course, depend upon the stage of the disease and the individual case. 
A decrease to one-half of the normal number may be seen in com- 
paratively mild cases; a million red cells is a common count. The 
number may fall to 500,000 and even lower. In one case reported 
by Quincke 1 a count of 143,000 was observed, and it is interesting 
to note that seventy-four days later the same patient had 1,234,000 
per cbmm. Osier 2 reports a case in which shortly before death the 
red cells fell below 100,000. This is the lowest count that has been 
recorded. In the stage of amelioration they may rise to 4,000,000 
and even higher. In the series collected by Strauss and Rohnstein 3 
1,240,000 was the average at the time when the patient first came 
under observation, and in Cabot's series of one hundred and ten 
cases the average number is almost identical — 1,200,000. 

In chlorosis, contrary to what is found in pernicious anemia, the 
red cells are usually not much diminished. In Cabot's 4 series of 
seventy-seven cases the average count was 4,050,000. At times, 
however, cases are met with in which the diminution of the red cells 
almost keeps step with the diminution in the amount of hemoglobin. 
Von Limbeck cites three cases with 1,750,000, 1,850,000, and 
1,930,000, respectively; and Hay em mentions an instance in which 
only 937,360 cells were counted. Such cases are exceptional. 

As in chlorosis so also in splenic anemia, the corpuscular anemia 
is of very moderate grade, even though the diminution in the amount 
of hemoglobin may be considerable. Of the forty-one cases collected 
by Osier, the average was 3,425,000; the lowest count was 2,187,000 
and the highest 5,200,000. 

A similar condition is found in Kala-azar, where the number of red 
cells is commonly reduced to 2,000,000 to 3,000,000. 

In leukemia the red cells are usually not diminished to a very 
great extent; and the oligocythemia is generally more marked in 
the lymphatic than in the myelogenous variety; the average figures 

1 Centralis, f. d. med. Wiss., 1877, No. 47; and Deutsch. Arch., 1877, vol xx. 

2 Johns Hopkins Hosp. Bull., 1902, vol. xiii, p. 251. 

3 Loc. cit. 

4 Clinical Examination of the Blood, Wm. Wood & Co. 



58 THE BLOOD 

in Cabot's series are 2,730,000 and 3,120,000, respectively. Counts 
of 1,000,000 or thereabout may, however, be met with. 

In pseudoleukemia the red cells may be only moderately dimin- 
ished, viz., between 3,000,000 and 4,000,000, but in some cases the 
corpuscular destruction is quite active, and in the last stages of the 
disease values may be found which are not much above 1,000,000 
or 1,500,000. 

The count which is obtained in post-hemorrhagic cases will depend 
very largely upon the amount of blood lost and the time at which 
the examination is made. The lowest counts, according to Lyon, 1 
Huhnerfauth, 2 and Siegel-Maydl, 3 are found between the second and 
the eleventh day. In Rieder's 4 case the figures varied between 
1,300,000 and 3,335,000; in those of Strauss and Rohnstein, 5 be- 
tween 1,119,000 and 4,420,000. A sudden reduction in the number 
to 1,000,000 or less is usually followed by a fatal result. 

In the anemias of infancy and early childhood the oligocythemia 
is often very pronounced. In the infantile pseudoleukemia of 
v. Jaksch especially low values may be found associated with an 
increase of the leukocytes of such extent that the ratio between 
the two may be suggestive of true leukemia; there is, however, no 
myelemia, but an increase of the normal types. In infantile leu- 
kemia of the lymphatic variety McCrae found 2,350,000 as the 
highest count. 

An extreme and rapidly progressive anemia is frequently noted in 
acute streptococcus infections. Grawitz 6 states that according to 
Rocher's investigations it is probable that the diminution of the red 
cells in septicemia is greater than in any other infectious disease and 
appears in a shorter time. Cases may indeed be encountered in 
which the question of pernicious anemia may enter into the diag- 
nosis, as occurred in two cases of gonorrheal endocarditis which 
were observed by Osier. 

An extreme grade of corpuscular destruction is also noted in 
malaria. In acute cases the loss of red cells during the first twenty- 
four hours may reach 1,000,000, and in two days even 2,000,000. 
In neglected chronic cases the count usually varies between 3,000,000 
and 4,000,000; the oligocythemia may, however, be far more ex- 
tensive, and Ewing 7 cites a case observed by Kelsch, with only 
583.000 red cells per cbmm. 

The anemia observed after typhoid fever is as a rule not very 
severe, but exceptional cases occur in which the loss of red corpus- 

1 Virchow's Archiv, 1881, vol. xciv. 

2 Ibid., vol. lxxvi. 

3 Wien. med. Jahrbuch., 1884. 

4 Beit. z. Kenntniss d. Leukocytose, Leipzig, 1892. 

5 Loc. cit. 

6 Klin, pathol. d. Blutes, Enslin, Berlin, 1902. 

7 On the Blood, Lea Bros, and Co., 1901. 



MICROSCOPIC EXAMINATION OF THE BLOOD 59 

cles is considerable. Osier cites an instance in which the number 
fell to 1,300,000. 

The post-rheumatic anemia is usually not so pronounced. 

In acute endocarditis Stengel 1 has noted a rapid decrease of the 
red corpuscles, often to 50 and even 40 per cent. 

In pulmonary tuberculosis the number of the red corpuscles runs 
a course parallel to that of the hemoglobin. Oligocythemia is really 
only seen during the third stage (2,000,000 to 2,500,000); while during 
the second stage, owing to an actual concentration of the blood 
(Grawitz), normal figures are the rule. In the first stage a diminution 
of their number (3,800,000) is only seen in patients who have repeat- 
edly suffered from tuberculous affections (scrofula) since childhood, 
and in whom the onset of the pulmonary disease has been gradual, 
while, on the other hand, normal values are found in individuals who 
appear to be in perfect physical health, who are well nourished, with" 
well-shaped chests, and without hereditary predisposition (Appel- 
baum). 2 

In acute gastritis, and usually in chronic gastritis also, the number 
of the red corpuscles is not diminished, while in carcinoma a marked 
oligocythemia occurs at some time in the course of the disease. In 
the earlier stages, however, this is often but little marked, and at 
times an apparent increase of the red cells is noted (relative poly- 
cythemia). Later a diminution is probably always found. In the 
severer forms of chronic gastritis a diminution is also fairly constant, 
but rarely so marked as in carcinoma, if we except those cases of 
gastric anadeny which present the clinical picture of a pernicious 
anemia. In the differential diagnosis between carcinoma of the 
stomach and pernicious anemia a count below 1,000,000 points to the 
latter disease. In ulcer of the stomach anemia of the chlorotic type 
is very common. In Cabot's series of 51 cases, 42 (80 per cent.) had 
a hemoglobin value of less than 50 per cent., and in the Hopkins series, 
reported by Futcher, the average value was 58 per cent., with a red 
count of 4,071,000. When hemorrhages have recently occurred the 
blood count may of course be very low. 

In the majority of cases of rickets there is no material diminution 
in the number of the red cells, while the hemoglobin may be much 
reduced, but in the severer forms with visceral complications there may 
be oligocythemia of extreme grade, v. Jaksch cites a case in which 
the red count fell from 1,600,000 to 750,000 within three months, 
and Luzet noted a drop to 500,000 within three weeks (Ewing). 

In congenital syphilis the oligocythemia is usually marked, except- 
ing in very mild cases, and in the severer infections the blood picture 
may simulate that of pernicious anemia. 

1 Loc. tit., p. 59. 

2 Berl. klin. merl. Woch., 1901, vol. xxxix, p. 7. 



60 THE BLOOD 

Behavior toward Aniline Dyes. Polychromatophiiia (Polychro- 
masia). — The normal living red cell possesses no affinity for dyes; it 
is achromatophilic. The normal fixed cell of the circulating blood, 
on the other hand, has a marked affinity for acid dyes, such as eosin, 
orange-G, acid fuchsin, etc.; it is accordingly said to be oxyphilic, 
and as it takes up only one color from a mixture of different dyes it 
is termed monochromatophilic. Under various pathological condi- 
tions which are associated with a marked grade of anemia cells are 
met with which are polychromatophilic. Such cells manifest an 
affinity not only for acid dyes, but simultaneously also for basic 
dyes, so that with a mixture of hematoxylin and eosin, or eosin and 
methylene blue, the red cells are not stained in the usual tint of the 
hemoglobin, but present a mixed Color in which the tint of the basic 
dye is more or less apparent (Plate III). 

As regards the significance of the polychromasia, Ehrlich main- 
tained that the condition was evidence of a degenerative process — 
of a coagulation necrosis of the discoplasm as a consequence of which 
this takes up albumins from the blood plasma, while it loses the 
power of holding its hemoglobin. The oxyphilia hence diminishes, 
while owing to the absorption of albumins a more or less well-marked 
basophilia develops. As a matter of fact polychromatophiiia is often 
seen in cells which are manifestly degenerating, and in myelogenous 
leukemia especially one frequently meets with nucleated red cor- 
puscles which are markedly polychromatic and in which the proto- 
plasm is evidently undergoing destruction, often appearing merely 
as a little hood attached to one side of the nucleus (see Plate III). 
Ehrlich accordingly speaks of an anemic or polychromatophilic 
degeneration of the blood. But, on the other hand, there is evidence 
to show that polychromasia may be the expression of a regenerative 
process, and we find as a matter of fact that the erythroblasts of the 
normal bone-marrow are for the most part polychromatophilic, and 
the more markedly so the younger they are. Megaloblasts are proba- 
bly always polychromatophilic (Plate III). Welker has shown 
that basophilic red cells are normally found in pigeons, mice, guinea- 
pigs, cats, and dogs, while they are absent in the horse and the ox. 
I have also found them in the blood of birds, reptiles, amphibia, and 
fishes. In those animals, moreover, in which the red cells of the 
circulating blood are normally nucleated a certain grade of poly- 
chromasia, according to my experience, appears to be the rule in all 
the younger cells; the pure hemoglobin tint is only found in the 
mature forms. 

Of late, Ehrlich has admitted the existence of a physiological 
polychromasia, but he still maintains that it may also occur as the 
expression of a degenerative process. 



PLATE III. 






,--m 







•..v.' 



. y - .. 



■I ,'^L:::. 



« 



™ 




<f 



W 



^^ 



i P 1 






LS. 



o, a group of red cells undergoing granular degeneration; 6, red cells showing Cabot's ring 
bocues ; c, normoblasts with nuclei undergoing karyolysis ; the bodies of the cells show granular 
degeneration ; d, normoblast with pyknotic nucleus ; f, red cell, suggesting loss of nucleus by extrusion- 
Q, red cell undergoing mitosis ; h, megaloblasts with polychromasia of protoplasm ; i, gigantoblast : 
/., young normoblasts, showing spoke-shape arrangement of the chromatin ; I, a group of plaques 



MICE SCO PIC EXAMINA TION OF THE BLOOD 6 i 

Literature. — Ehrlich, Charite Annalen, vol. x, p. 136. Engel, Deutsch. med. 
Woch., 1899, p. 209. Gabritsehewsky, Arch. f. exp. Path., vol. xxviii, p. 83; Zeit. 
f. klin. Med., vol. xxvii, p. 492. Askanazy, ibid., vol. xxi, p. 415. Maragliano 
and Castellino, ibid., vol. xxi, p. 415. 

Diabetic Chromatophilia. — Bremer has pointed out that a distinct 
difference exists in the affinity of diabetic blood for certain aniline 
dyes, as compared with non-diabetic blood. For, whereas non- 
diabetic blood is readily stained with Congo-red, methyl blue, eosin, 
etc., diabetic blood is distinctly refractory, while such dyes as Biebrich 
scarlet, which readily stain the diabetic blood, do not color non- 
diabetic blood. 

Regarding the nature of the substance in diabetic blood which is 
responsible for this peculiar behavior, little is known, but it appears 
certain that the reaction is not dependent upon the presence of glu- 
cose nor upon the degree of alkalinity of the blood, as suggested 
by Lepine and Lyonnet. Bremer's claim that the reaction is pathog- 
nomonic of diabetes and glucosuria and may even yield positive 
results in the pre-diabetic stage of the disease, and when the sugar 
has temporarily disappeared from the urine, has been confirmed in 
all essential points, both in this country and abroad. A few inter- 
esting exceptions, however, have been noted. In animals, for 
example, in which glucosuria has been artificially produced by 
means of phlorhizin, the reaction does not occur, whereas in phloro- 
glucin-diabetes positive results are obtained. In Bremer's entire 
series of diabetic cases a negative result was obtained but once, and 
in this instance he believes that the diabetes was of the renal type, 
and analogous to the phlorhizin-diabetes of animals. He suggests 
that it may thus be possible to differentiate this form from the hema- 
togenic variety, using the latter term in its widest sense. Lepine 
and Lyonnet report a positive result in one case of leukemia, but 
Bremer believes this to have been due to faulty technique. Hartwig 
finds that Bremer's reaction is constant in diabetes, but that it may 
also occur at times in other conditions. 

The description of Bremer's diabetic blood test is omitted at this 
place, as it has not proved practical for routine work. 

For a consideration of the technique see the Literature below. 

Literature. — L. Bremer, " An Improved Method of Diagnosticating Diabetes 
from a Drop of Blood/' N.Y. Med. Jour., 1896; Centralbl. f. inn. Med., 1897, p. 521. 
lie Goff, React, chrom. du sang diabet., Paris, 1897. Lepine and Lyonnet, Lyon 
med., vol. lxxxii, p. 187. Hartwig, Deutsch. Arch. f. klin. Med., vol. lxii, p. 287. 

Granular Degeneration of the Red Cells.— Under pathological 
conditions red cells may be met with which contain basophilic gran- 
ules. These are readily stained with methylene blue, methylene azure, 
thionin, etc. Methyl green, however, which is a specific unclear 
dye, does not stain the granules. Their size, form, and number 



62 THE BLOOD 

are variable. While the majority are round, others are rod-shaped 
or biscuit-shaped. The largest granules are found in pernicious 
anemia and in cases of lead poisoning with intestinal manifes- 
tations. They are then quite readily seen and attract attention at 
once (Plate III). In most other diseases in which they occur they 
are much smaller, and on superficial examination they may indeed 
be overlooked; some cells at first sight merely look a little off-color, 
and it is seen only on very careful examination that the apparent 
polychromasia is in reality due to the presence of large numbers of 
minute dots. Very often, in especially anemic cells, the granules 
are arranged in the peripheral portion of the cell. Their number 
is exceedingly variable; generally speaking, it depends upon their 
size; when they are especially large they are relatively less numerous. 

The granules may occur in cells of normal size or color, in poiki- 
locytes, and in nucleated ied cells, both of the normoblastic and the 
megaloblastic type, especially the former. Not infrequently they 
are seen in cells which are markedly polychromatic, but, like Grawitz, 
I do not believe that granular degeneration represents a phase of 
polychromasia. 

In disease they are most constant and numerous in pernicious 
anemia, in lead poisoning, and in malaria; they are less constant 
and less numerous in the leukemias, in pseudoleukemia, in the 
cachexias referable to septic infection, syphilis, carcinomatosis, and 
in the final stages of tuberculosis. In chlorosis and in the anemia 
of chronic nephritis they are absent; in two cases of v. Jaksch's 
anemia, in which nucleated red cells were quite numerous, I ob- 
tained negative results. 

In pernicious anemia granule cells are frequently found in the 
interval and at a time when the blood picture is otherwise practically 
normal. I have seen them most numerous in a case in which blood 
crises occurred from time to time (see page 60); almost every nor- 
moblast contained granules; non-nucleated granule, cells were how- 
ever, at the same time present in large numbers Late in the disease, 
or in aplastic pernicious anemia, granule cells in my experience 
may be absent. 

In lead poisoning granule cells are practically found without 
exception, and may be encountered at a time when no clinical symp- 
toms are manifest. The amount of lead which is necessary to call 
forth their appearance is quite small, and it is a common experience 
to meet with a small number after the administration of lead in 
medicinal doses. I have found them after the ingestion of only 0.5 
gram given in divided doses in the course of forty-eight hours. In 
cases of lead poisoning they persist for a long time after exposure 
has ceased. In one case of double wrist- and ankle-drop I could 
still demonstrate granule cells after five months. 

In malaria granule cells are also common. Plehn found them 



MICROSCOPIC EXAMINATION OF TEE BLOOD 63 

in Europeans after a short sojourn in the tropics, and looked upon 
the granules as spores of the malarial parasite. 

In septic cases and in the cachexia of carcinomatosis they are not 
numerous; in a case of cancer of the stomach with only 27 per cent, of 
hemoglobin, which I recently observed, I found no granule cells. 

In the early stages of phthisis granular degeneration is not seen, 
but it may occur later, when a general septicemia has supervened. 

Takasu 1 notes the occurrence of granule cells in infants affected 
with beriberi, especially in the acute severe cases. The same appar- 
ently occurs in the adult. 

As regards the significance of the granules, Engel, Ehrlich, and 
others have suggested that they are most likely products of karyor- 
rhexis. Others maintain, and I think rightly so, that they are not 
of nuclear origin. They may be found at a time when not a single 
nucleated red cell is demonstrable in the blood and nucleated red cells 
may be seen in which no sign of karyorrhexis is manifest, while the 
body of the cell is studded with granules. They may be found in 
nucleated cells which are undergoing karyokinetic division. Unlike 
the nuclei of the erythroblasts, the granules have no affinity for 
methyl green, which is a specific nuclear dye. This can be shown 
very well by staining with methyl-green-pyronin, when granular 
products derived from nuclei are stained green, while the stippling 
in the same cell appears red. A few observers claim to have stained 
the granules with methyl green; this merely shows that their dyes 
were contaminated with methylene blue. 

According to Grawitz and others granule cells are not commonly 
found in the bone-marrow even when they are numerous in the cir- 
culating blood; when they do occur, they are not more numerous 
than in the peripheral vessels. Grawitz hence regards their presence 
as an indication of a degenerative change in the hemoglobin, and 
speaks of the phenomenon as "granular degeneration." Others 
regard the bone-marrow as their place of formation. Nageli 2 thus 
comes to the conclusion that they are formed in the bone-marrow, 
because they only appear in artificial lead intoxication, when this 
is continuously established, and disappear when larger doses are given. 
Preceding the death of the animal they are not found. Opposed to 
the peripheral formation of the granules and Grawitz's degeneration 
hypothesis is the occurrence of granule cells in the blood of embryos. 

According to Pappenheim stippling is not found in erythroblasts in 
the bone-marrow under normal conditions, but only when there is 
excessive regeneration, as in the embryo, in pernicious hemolytic 
anemia, in myelophthisic neoplastic anemia, in myelogenous pseudo- 
leukemia and lymphadenoid leukemia and lymphosarcomatosis of the 
bone-marrow. Schmauch has observed similar appearances in the 

1 Folia hsemat., vol. i, p. 501. 2 Munch, med. Woch., 1904. 



64 THE BLOOD 

blood of healthy cats, and Engel has described the occurrence of 
granule cells in the blood of early cat embryos. I have found granule 
cells in the blood of various animals and occasionally one meets 
with an isolated cell in apparently normal individuals. 

Whether or not the granule cells which Vaughan 1 has demonstrated 
in normal wet specimens with Unna's polychrome methylene blue 
are identical with the variety described above is not certain. Their 
number varied quite constantly between 1.8 and 5 per cent. The 
examinations were conducted with wet blood, a drop of the staining 
fluid being placed upon the site of the puncture. At first the granules 
are red, but after some time they change through a purple to a pro- 
nounced bluish. Positive results were also obtained under various 
pathological conditions, especially in pernicious anemia, where their 
number was about ten times as great as in normal blood. In new- 
born infants they average 4.7 per cent. Vaughan regards the gran- 
ules as nuclear remains and states that he rarely found stippling and 
nuclei in the same cell. In my own experience normoblasts in 
pernicious anemia are very frequently granular (see Plate III). 
Analogous results have been obtained by Cadwalader. 2 

Not to be confounded with "granular degeneration" is the stippling 
of Schiiffner, 3 Ruge, 4 and Goldhorn, which is seen in many red cells 
infected with tertian parasites. This is brought out with methylene 
azure and may also hide the parasite from view. 

Literature. — E. Grawitz, '-Ueber kornigeDegeneration d. rothen Blutzellen," 
Deutsch. med. Woch., 1899, No. 36, p. 585; "Klinische Bedeutung u. experiment. 
Erzeugung korniger Degenerationen," etc., Berlin, klin. Woch., 1900, p. 181 ; 
" Granular Degeneration of the Erythrocytes, " etc., Amer. Jour. Med. Sci., 1900, 
vol. cxx, p. 277. Bloch, Deutsch. med. Woch., 1899, V. B. p. 279. Litten, ibid., 
No. 44. Behrendt, ibid., No. 44. White and Pepper, " Granular Degeneration 
of the Erythrocyte," Amer. Jour. Med. Sci., 1901, vol. cxxii, p. 266. C. E. Simon, 
International Clinics, 1902, vol. i, p. 69. Stengel, White, and Pepper, Amer. Jour. 
Med. Sci., 1902, vol. cxxiii, p. 873. 

Cabot's Ring Bodies. — Cabot has drawn attention to the occa- 
sional occurrence in red cells of curious ring bodies which are usually 
stained red with Wright's modification of Leishman's stain, but 
which may also take on a blue color. He found such rings in per- 
nicious anemia, in lead poisoning, and in lymphatic leukemia. I 
have been able to demonstrate the same structures with the eosinate 
of methylene blue, and could verify Cabot's observation that they 
occur in granule cells, but may also be found in apparently normal 
red corpuscles (Plate III). No doubt they bear some relation to the 
nucleoids. (See Fig. 15.) 

Literature. — Cabot, Jour. Med. Research, 1903, vol. ix. 

1 Jour. med. Res., December, 1903. 

2 Amer. Jour., Februarv, 1905, p. 213. 

3 Deutsch. Arch. f. klimMed., 1899, vol. lxiv, p. 428. 

4 Zeit. f. Hyg. und Infectkrht., 1900, vol. xxxiii, p. 178. 



MICROSCOPIC EXAMINATION OF THE BLOOD 



65 



Ehrlich's Hemoeglobinemic Innenkbrper. — These structures may 
be encountered in red cells in conditions associated with extensive 
hemocytolysis the result of specific blood poisons. The individual 
body is round and characterized by its affinity for acid dyes. 




Fig. 15. —Cabot's ring bodies. 

Nucleated Red Corpuscles. Erythro blasts. — Nucleated red cor- 
puscles are not found in the circulating blood of normal individuals, 
excepting at birth and during the first days of life, when it is not 
unusual to meet with an occasional cell of this type. In the bone- 
marrow, however, they are always found. It is here possible to dis- 
tinguish two types, viz., the normoblast and the megaloblast. The 
latter is ontogenetically the older and gives rise to the normoblast 
through a process of homoplastic differentiation by cell division; 
it thus bears the same relation to the normoblast which exists between 
the large lymphocyte and the small lymphocyte, and the amblychro- 
matic myelocyte and the trachychromatic myelocyte (which see). 
The megaloblast itself results from the large lymphocyte through 
direct heteroplastic transformation and ages into the macrocyte, 
while the normoblast similarly develops into the normocyte. (See 
schema on p. 72.) 

While at a certain period of embryonic life megaloblastic blood 
corpuscle formation plays a prominent role, megaloblasts are found 
only in small numbers in the bone-marrow of the normal adult. 
Normoblasts, on the other hand, are numerous and control the usual 
red corpuscle production exclusively. 

The Normoblasts. — The normoblasts (see Plate III), like the 
normal red cells of the circulating blood, have a diameter which 
5 



66 THE BLOOD 

varies from 6 to 9 fx. The nucleus in the youngest cells occupies 
a central position, and is larger and relatively poorer in chromatin 
than in the older cells, where it is frequently located eccentrically. 
The size varies between 2 and 4 //. The appearance of the normo- 
blast in the peripheral circulation is variable (Plate III). In most 
cases young cells are seen with a radiary arrangement of the chro- 
matin and polychromatophilic protoplasm. At other times older 
cells with densely staining pyknotic nuclei and oxyphilic protoplasm 
are encountered and again we may meet with cells in which manifest 
karyolysis is going on, as evidenced by budding of the nucleus and 
diminished chromatophilia. Fragmentation of the nucleus (karyor- 
rhexis) may also be seen, as also free nuclei as such. Mitoses are not 
uncommon in pernicious anemia and leukemia. 1 

In the majority of cases in which normoblasts are found in the 
blood these are well preserved, but in myeloid leukemia more 
especially it is common to meet with cells in which the protoplasm 
surrounding the nucleus is much diminished in amount and presents 
a ragged outline. These cells are manifestly degenerating, and in 
many specimens the protoplasm will be seen reduced to a little 
hood which is attached to one side of the nucleus (Plate III). 
Such cells in my experience are always polychromatophilic and are 
apt to be mistaken by the beginner for lymphocytes. 

The occurrence of normoblasts in the circulating blood is always 
evidence of stimulation of the bone-marrow, which may occur either 
indirectly, as the result of an "anemic" condition of the blood (second- 
ary myelopathy), or directly, as in disease of the bone-marrow per se 
(primary myelopathy). We may accordingly meet with normo- 
blasts in almost any form of anemia, be this the result of traumatism 
(posthemorrhagic), of inanition, or of organic disease. 

In the acute forms of anemia they are apt to be most numerous, but 
even in the more chronic cases and in cachectic conditions specimens of 
blood may be obtained in which one or more normoblasts are seen in 
every field. In the secondary anemias, however, they are less common. 

In active cases of pernicious anemia and the different leukemias 
normoblasts are quite constantly met with in fairly large numbers. 
Their continued absence in pernicious anemia is usually evidence of 
an aplastic condition of the bone-marrow and a bad omen. 

At times there occur sudden invasions of the circulating blood 
by red cells, many of which are nucleated; this phenomenon v. 
Noorden terms a blood crisis, and it is noteworthy that the invasion 
of the red cells may be preceded and accompanied by a very ex- 
tensive increase of the leukocytes. Ehrlich cites a case of hemor- 
rhagic anemia, reported by v. Noorden, in which at the time of such 
a blood crisis the normoblast were so numerous, while hyperleuko- 

1 G. Dock/' Mitosis in Circulating Blood," Trans. Assoc. Amer. Phys., 1902, p. 484. 



MICROSCOPIC EXAMINATION OF THE BLOOD 67 

cytosis of a high grade existed at the same time, that the blood con- 
dition strongly suggested the existence of a myeloid leukemia. The 
increase of the red cells in this case amounted to almost double their 
original number. 

To estimate the extent of a blood crisis, the following examina- 
tions are necessary: 

(a) A determination of the absolute number of red corpuscles. 

(6) A determination of the ratio between the white and red cells. 

(c) A determination of the ratio between the nucleated red and white 
cells. 

Example. — Supposing that in a given case 3,500,000 red corpus- 
cles are found in the cbmm., while the ratio of the white to the red 
corpuscles is 1 to 100, and that of the nucleated red to the white 1 to 
100; 3500 nucleated red corpuscles must hence be present in each 
cbmm. of blood — i. e., 1 for each 1000 of normal red corpuscles. 

The Megaloblasts. — These are usually from two to three times as 
large as the normoblasts, and may attain even more extensive propor- 
tions (Ehrlich's gigantoblasts). (See Plate III.) But some specimens 
are only a very little if at all larger than the common red cells ; these 
probably represent young daughter cells. The megaloblasts are 
provided with a relatively large centrally located nucleus, which is 
wide-meshed and which with the triacid stain is not colored nearly 
so deeply as the normoblastic nucleus. In some specimens, indeed, 
the affinity for methyl green is so little marked that at first sight a 
nucleus can hardly be distinguished. With those staining mix- 
tures, on the other hand, which contain methylene blue as base, it 
can always be fairly well made out. But owing to the fact that these 
cells are almost invariably polychromatophilic, the nucleus may at 
first be overlooked, as the ploychromatic protoplasm appears in the 
meshes of the nucleus and sometimes differs but little in color from 
the chromatin. The inexperienced not infrequently mistake such 
cells for large mononuclear leukocytes that are somewhat off-color; 
the character of the nucleus, however, viz., its wide meshwork, 
should prevent this mistake. 

Mitoses in megaloblasts are at times seen. 

As already mentioned the megaloblast is essentially a cell of 
embryonic life. After birth, under normal conditions a few megalo- 
blasts may be found in the blood of very young infants, and it is 
noteworthy that in the severe types of secondary anemia megalo- 
blasts are far more apt to occur in children than in adults. But 
even then they are rare. In the bone-marrow of the adult they are 
present in very small numbers. According to Ehrlich, the presence 
of megaloblasts in the blood is evidence of a reversion of blood 
formation to the embryonic type and of grave prognostic import. 
He regarded their presence as indicative of essential pernicious 
anemia- and, as a matter of fact, they are here quite constantly 



68 THE BLOOD 

met with and represent one of the most important features of the 
disease. They are rarely numerous, however, and there are cases 
in which they are absent 1 (aplastic anemia). 

The modern tendency is to regard the appearance of megaloblasts 
in the blood as evidence of an anemia of unusual severity, viz., as 
a degenerative-regenerative symptom, and not as an indication of 
any one disease. While they are undoubtedly most constant in per- 
nicious anemia, they may also be met with in other forms. They 
have been found in leukemia, in the pseudoleukemia of infants, in 
lead poisoning, and, even in chlorosis, and as I have pointed out already, 
in some of the severe types of secondary anemia occurring in young 
children. In cancer of the stomach, according to Osier and McCrae, 
they are rarely if ever found. Askanazy 2 has reported an interesting 
case of bothriocephalus infection in which the megaloblastic type of 
blood regeneration disappeared after expulsion of the parasites — 
sixty-seven in number — and was replaced by the normoblastic type, 
the case ending in recovery. 

The appearance of megaloblasts in extra-uterine life merely in- 
dicates an incomplete maturation of young elements, their consump- 
tion and consequent increased production. The following sketch, 
taken from Pappenheim, gives an idea of the relation of normoblasts 
and megaloblasts to the different types of anemia: 

Under normal conditions Pappenheim's large lymphocyte (see 
schema, p. 72) gives rise to the young megaloblast, which in turn dif- 
ferentiates itself at once into young normoblasts. The young normo- 
blast ages to the pyknotic normoblast and loses its basophilic nuclein 
as a result of chemical karvolysis. In this manner an apparently 
non-nucleated erythrocyte results, which loses its nucleoid later, in the 
blood , as blood platelet in consequence of variations in the tonicity of 
the plasma. In severe toxogenic anemias, on the other hand, there is 
an arrest of development upon an embryonic basis. A certain propor- 
tion of young megaloblasts multiplies homoplastically ; another portion 
matures to old megaloblasts, while a third fraction only becomes 
differentiated to young normoblasts. Of these in turn one portion 
matures to the old forms, which dislodge their nuclei in the anemic 
serum in toto, while another portion loses the nucleus during the 
process of hastened maturation by karyorrhexis. As a consequence 
many of the anemic normocytes contain no nucleoids, and the blood 
as a consequence contains only small numbers of blood platelets. 

Pyknotic normoblasts, as also young megaloblasts (of the type of 
the large lymphocyte), may thus be encountered in all forms of severe 
anemia of whatever origin. In the kryptogenetic type of pernicious 
anemia and bothriocephalus anemia, however, old megaloblasts (of the 

1 Pane, " SmT anemia progressiva mortale senza corpuscoli rossi, nucleati nel 
sangue," Riform. med., 1900, No. 263. 

2 Zeit. f. klin. Med., 1895, vol. xxvii, 



MICROSCOPIC EXAMINATION OF TEE BLOOD 69 

type of the large mononuclear leukocyte) are further seen, as also 
young normoblasts (of the type of the small lymphocyte) under- 
going karyorrhexis. 

Generally speaking the number of erythroblasts is no indication of 
the severity of the case, but merely indicates the extent to which the 
bone-marrow responds to the blood destruction. The appearance of 
megaloblasts is- hence not necessarily an absolutely unfavorable 
symptom, but simply the expression of an unusually high activity of 
the erythropoietic tissue. 

In cases of traumatic anemia unusually small nucleated red cells 
have at times been observed. These are termed microblasts. They 
have attracted but little attention and are quite rare. I have seen 
such cells, measuring not more than 3 to 3.5 ft, in a case of pernicious 
anemia at the time of the blood crisis, when large numbers of normo- 
blasts were also present. 

The Leukocytes. 

General Characteristics. — The leukocytes, or white corpuscles 
of the blood, as seen in the wet preparation (Plate II), are roundish 
or irregularly shaped cells, which very in size, but for the most part 
are larger than the red corpuscles. They are all nucleated, and, as 
the term indicates, devoid of coloring matter. In a general way they 
may be divided into two distinct classes, viz., those which are granular 
and those which are not granular. 

The granular cells (granulocytes) are by far the most numerous, 
and are characterized by the fact that they are capable of active loco- 
motion. Even without a warm stage it is almost always possible to 
observe this in the ordinary wet preparation. The moving cells at 
once attract attention by their irregular outline. On careful exami- 
nation with a high power it will be noted that the cell advances in a 
definite manner, which is quite analogous to what is seen in the ameba. 
The protoplasmic portion manifestly consists of two parts, viz., a non- 
granular hyaline ectosarc and a granular endosarc. As the leukocyte 
progresses the hyaline ectosarc advances with a flowing motion, form- 
ing a distinct layer in front of the granular endosarc, which itself then 
merges into the non-granular portion. The moving leukocyte is 
roughly pear-shaped, with the base in advance, while the rear end 
tapers markedly and frequently seems to drag behind it a small, 
roundish mass which, like the main body of the cell, is also granular. 
These granular leukocytes are true "phagocytes and take up foreign 
matter into their interior like amebas. According to MetchnikofT, the 
phagocytic function is the most important function of the leukocytes, 
and the outcome of a bacterial invasion, figuratively speaking, will 
depend upon the superiority of the organisms engaged in warfare. 

The nucleus of the granular leukocytes is either polymorphous — 
i. e., it is composed of different lobes which are joined together — or 



70 THE BLOOD 

it may be multiple. Such cells are hence spoken of as polymorpho- 
nuclear and polynuclear leukocytes, respectively. The polymorphous 
cells represent an earlier stage in the development of the polynuclear 
cell. 

While the granules in the majority of the leukocytes are fine (Plate 
II), on careful search some cells will be found in which they are 
coarse and highly refractive. This coarsely granular variety is very 
characteristic in appearance and at once attracts attention. The 
cells are far less numerous, however, and, as a matter of fact, represent 
only from 1 to 4 per cent, of the total number of the leukocytes, while 
the finely granular variety represents from 60 to 70 per cent. 

The non-granular leukocytes, in contradistinction to the granular 
variety, are mononuclear, with very little tendency to polymorphism. 
They are quite hyaline in appearance, and are readily overlooked by 
the beginner unless a somewhat subdued light is used in the exami- 
nation. Two varieties may be recognized : one about the size of a red 
corpuscle, the other somewhat larger. The nucleus in both varieties 
occupies a considerable portion of the cell and is surrounded by a 
layer of protoplasm which is practically hyaline. Every cell, it is 
true, contains a few granules collected at a certain point along the 
periphery, where the protoplasm is more extensively developed than 
elsewhere; but these granules, in contradistinction to those which we 
see in the polynuclear varieties, probably represent nodal points in the 
cytoreticulum, and not a specific secretory product, as which Ehrlich 
and his school view the granules of the polynuclear variety. In the 
small mononuclear form one or sometimes two small, brownish 
granules can usually be discerned somewhere in the peripheral layer 
of the protoplasm. Of the significance of this granule, so far as I 
am aware, nothing is known, nor has its presence been previously 
described (Plate II). 

The non-granular mononuclaer leukocytes, in contradistinction to 
the polynuclear granular variety, were formerly regarded as non- 
motile. Jolly, Wolff, and others have shown, however, that they also 
are capable of changing their form even though progressive loco- 
motion may not occur. The change in form can readily be demon- 
strated even without a warm stage, and it will be observed that the 
nucleus takes an active part in these changes. 

Classification. — While it is possible to distinguish the different 
varieties of leukocytes in the wet and unstained preparation, a more 
complete picture of the structure of the individual forms may be 
obtained from a study of stained preparations. The study of such 
preparations, moreover, forms the most satisfactory basis for the 
classification of the different forms. We distinguish the following 
varieties : 

1. The Lymphocytes (Small Mononuclaer Leukocytes, or Micro- 
lymphocytes) (Plate IV). — The lymphocytes which occur normal] v 



PLATE IV. 











Lymphocytes. 

'he cell a shows nucleus after division, each with a nucleolus ; b, a plasma 
(irritation form, ptilogocyte). 



MICROSCOPIC EXAMINATION OF THE BLOOD 71 

in the blood are for the most part a little smaller than the red corpuscles 
or of equal size. The nucleus is single and surrounded by a narrow 
rim of protoplasm which is generally described as non-granular; 
but, as I have pointed out, a few granules can almost always be made 
out in the wet preparation at a certain point along the periphery, 
where the protoplasm is a little more extensively developed. These 
granules, however, probably represent nodal points of the cytoreticu- 
lum, and are not to be regarded as in any way analogous to the granules 
which are met with in the polynuclear leukocytes. Nucleus and pro- 
toplasm are both basophilic, and, generally speaking, the protoplasm 
is so more markedly than the nuclus. This is best seen in specimens 
that have been stained with a methylene-blue mixture, where the lym- 
phocytes for the most part present a comparatively feebly staining 
nucleus which is surrounded by a rim of dark blue. Other cells 
belonging to the same group, however, will also be seen in which this 
is not marked, but in which the staining affinities of both nucleus and 
protoplasm appear about the same or in which the protoplasm may 
even be lighter in color. These cells are generally a little larger than 
the first variety, with a somewhat broader zone of protoplasm and an 
eccentric position of the nucleus. They represent a later stage in the 
development of the deeply staining cell, and are sometimes termed 
medium-sized lymphocytes. A still larger form may also be met with, 
but is rarely seen under normal conditions. The staining properties 
of these large lymphocytes (macrolymphocytes) are essentially the same 
as those of the smaller varieties. The position of the nucleus may be 
either concentric or eccentric, as in the smaller forms, and a nucleolus 
is frequently demonstrable. This large type is notably seen in acute 
lymphatic leukemia, where it is usually the predominating cell. In 
smaller numbers it is also found under other pathological conditions 
which are associated with a hyperplasia of the lymphadenoid 
tissue. 

According to Pappenheim, the large lymphocyte represents the 
ancestral cell (Ur or Stammzelle), from which all other leukocytes, as 
well as the red cells, aie indirectly derived as the result of heteroplastic 
differentiation. (See schema, p. 72.) The large lymphocytes 
are identical with Benda's lymphogonia, Troje's lymphoid marrow 
cells, Nageli's myeloblasts and the undifferentiated lymphoid cell of 
Michaelis, Wolff, and Turck. 

With certain dyes, like methylene blue, the protoplasm of the 
lymphocytes does not appear perfectly homogeneous, but presents a 
peculiar granular appearance. This is referable to nodal points of 
the cytoreticulum and does not represent a true granulation. With 
methyl green, and hence with Ehrlich's triacid stain, the protoplasm 
is perfectly homogeneous and appears as a pale rim about the some- • 
what more deeply staining nucleus. While it is thus impossible 
with the usual dyes to demonstrate the existence of a true granula- 



72 THE BLOOD 

tion in the lymphocytes, Michaelis 1 has called attention to the fact 
that with eosin-methylene-azure solutions (p. 132) distinct granules 
can be seen (azurophilic granules). Their significance, however, 
has not been established. Very curiously these granules could not be 
demonstrated in the lymphocytes obtained from the lymph glands 
directly, and it appears that they are present in only a certain percent- 
age of those occurring in the blood. The number of granules in a cell 
is variable; in some only two or three are seen, while in others the 
protoplasm is literally studded with them. Their size varies between 
that of the common neutrophilic and that of the eosinophilic varieties 
(Plate V). 

INTERRELATION OF LEUCOCYTES AND ERYTHROCYTES 

» = ' 

ERYTHROBLASTS LYMPHOCYTES GRANULOCYTES 



Large lymphocyte — * Large mononuclear — =>■ Transition form 



Megaloeyte « — Old megaloblast < — Young megaloblast 



n 



Normocyte <= — Old normoblast <= — Young normoblast 



Young amblychromatic — ^Older form — > Mature form 
myelocyte a,J,€ <*,"/,€ Cl,7,e 



Tracliychromatic — =>Po1ymphorpho — »Polynuelear 
myelocyte 0., "y,€ nuclear leucocyte leucocyte 

ay.e a, y.e 



Young small lymphocyte — >01dor form — »Rieder's form 



> Direct cytogenetic development ' * Homoplastic differentiation by cell divisiox 

> — > Direct heteroplastic transformation 

In wet specimens, as I have pointed out, one or two reddish- 
brown granules are quite commonly seen in most of the lymphocytes. 
In stained preparations these cannot be demonstrated. 

The outline of the cell in the smaller forms is usually fairly smooth, 
but in the larger varieties it is often shaggy, and at times specimens 
are seen with a number of distinct knobs. 

The nucleus, in the smaller forms especially, is concentrically 
located, while in the larger varieties, in which the protoplasm is 
more extensively developed, it commonly occupies an eccentric 
position. In the stained specimens, especially in the larger cells, it 
is sometmies surrounded by a faint areola, which is probably owing 
to artificial retraction. The nucleus is more commonly oval or bean- 
shaped than round; deep invaginations are not often seen and frag- 
mentation of the nucleus is rare. Such cells present an appearance 
which is altogether different from that of the true polynuclear elements. 

Lymphocytes undergoing mitosis are sometimes seen in the blood 
of lymphatic leukemia. Characteristic figures, however, are com- 
paratively rare, and it is more common to meet with cells in which 

1 Michaelis and Wolff, Virchow's Archiv, 1902, vol. clxvii, p. 151. 



PLATE V, 



# 




« 





e i 










€ 






O 




Leukocytes. 

a, microlymphocytes; a 1 , same, showing azurophilic granules; ft, large mononuclear leukocytes 
c, neutrophilic polymorphonuclear elements, d, adult eosinophile; e, neutrophilic myelocytes 
/. eosinophilic myelocyte: g, mast-cell; h, karyokinetic normoblast. Stained with Wright's stain. 



MICROSCOPIC EXAMINATION OF THE BLOOD 73 

division of the nucleus has already occurred (Plate IV). In hema- 
toxylin-eosin specimens it is usually possible to demonstrate a nu- 
cleolus, but in eosin-methylene-blue preparations my experience 
has been that they are not usually seen in the lymphocytes of the 
normal blood, and seem to be comparatively infrequent also in the 
blood of lymphatic leukemia. Occasionally, however, specimens are 
met with in which they are distinct, and at the same time multiple; 
in such cases active cell division seems to take place in the circulating 
blood. 

In adults the number of the lymphocytes normally varies between 
20 and 30 per cent. Higher values are found in young children, 
especially during the first year of life, when the lymphocytes con- 
stitute from 50 to 60 per cent, of the total number. At birth, how- 
ever, they are less numerous than in adult life, viz., only about 15 to 
16 per cent. Later they increase and by the twelfth day it is usual to 
have from 40 to 50 per cent. After the fifth year adult values are 
normally the rule. 

In disease the number of the lymphocytes may be increased or 
diminished, conditions which are spoken of respectively as lympho- 
cytosis and lymphopenia. 

While it was formerly supposed that the lymphocytes originate 
only in the lymph glands proper, there is evidence that they may 
be formed wherever there is lymphoid tissue, and hence also in the 
spleen and in the bone-marrow. They are probably derived from 
the large lymphocytes of the germinal centres indirectly through a 
process of differentiating karyokinesis, and represent fully differ- 
entiated cells which are incapable of further development. 

2. The Large Mononuclear Leukocytes (Splenocytes) . — These are 
mostly two or three times as large as the red corpuscles and pro- 
vided with a large single nucleus, which is surrounded by a relatively 
wide zone of non-granular protoplasm (Plate V). The nucleus in 
some cells is oval or elliptical, while in others it is more or less invagi- 
nated (Ehrlich's transition forms). 

In the wet preparation the large mononuclear leukocytes are 
exceedingly hyaline, so that they are readily overlooked by the 
beginner. Both nucleus and protoplasm are basophilic, but much 
less markedly so than in the lymphocytes, and it is noteworthy that 
the protoplasm usually possesses a less marked affinity for the basic 
dye than the nucleus. Cells are also met with, however, in which 
the affinity for the dye is about the same in both. If by chance this 
occurs in specimens which are somewhat smaller than usual, a certain 
amount of difficulty arises in differentiating such small " large" mono- 
nuclear leukocytes from the older lymphocytes. A hard-and-fast 
line of distinction cannot here be drawn, and in every differential 
leukocyte count the personal equation will of necessity enter into 
consideration. The salient characteristics of the two types should, 



74 THE BLOOD 

however, be borne in mind; in the lymphocytes the protoplasm is 
but feebly developed in relation to the size of the nucleus, while in 
the large mononuclear leukocyte the reverse is true. The proto- 
plasm in the latter, moreover, is apparently much more delicate in 
structure, and is readily wrinkled by contact with adjacent cells; 
not infrequently cells of this type are found which have manifestly 
been torn or otherwise injured during the preparation of the speci- 
men; the lymphocytes, on the other hand, are usually well-preserved 
and clear-cut, sharply defined cells. 

In preparations that have been stained with Ehrlich's triacid both 
nucleus and protoplasm are very faintly colored and the latter appears 
perfectly homogeneous; but in specimens which have been stained 
with mixtures containing methylene blue as the basic component, 
the protoplasm presents a somewhat granular appearance, which, as 
in the lymphocytes, is referable to the existence of a cytoreticulum. 
A certain proportion of the large mononuclear leukocytes (including 
the transition forms), as in the case of the lymphocytes, also contain 
azurophilic granules. 

Inclusive of the transition forms the large mononuclear leukocytes 
normally represent from 1 to 6 per cent, of the total number. They 
are relatively more numerous in young children, in whom the highest 
values are found between the sixth and ninth day after birth. Many 
of the cells at this time are of the type of the transition form; they 
may number 18 per cent.; but even in older children one commonly 
finds a larger proportion of these cells than in adults. 

According to Pappenheim the large mononuclear leukocytes develop 
directly, cytogenetically, from the " large" lymphocytes, and then age 
into the "transition forms" which represent the final stage in the 
development of this type. The former view, according to which the 
large mononuclear leukocyte develops directly cytogenetically from the 
small lymphocyte and later ages into the polynuclear neutrophile, has 
been abandoned. 

For the most part the large mononuclear leukocytes develop in the 
spleen (hence the term splenocytes). 

3. The Neutrophilic Polynuclear Leukocytes (Plate VI). — These 
cells are a little smaller than the large mononuclear leukocytes and 
represent the finely granular variety already mentioned. They are 
active phagocytes and as such capable of progressive locomotion. 
The nucleus in the younger cells is polymorphous, while the older 
cells are actually polynuclear, the number of lobes varying from two 
to six. In stained specimens the nucleus shows a coarsely reticular 
structure with nodal thickenings and is very markedly basophilic. 
The protoplasm, on the other hand, is very feebly oxyphilic. 

Embedded in the protoplasm are numerous fine granules — the 
^-granulation of Ehrlich — which are characterized by their affinity 
for neutral dyes. Hence the term polynuclear neutrophilic leukocytes. 



PLATE VI. 













Granulocytes. 



a, polynuclear neutrophilic leukocytes; b, polynuclear eosinophilic leukocytes; c, mast-cells; 
d, young eosinophilic myelocytes; e, neutrophilic myelocytes; /, the nucleus here has just undergone 
division; the clear space is a vacuole. 



MICROSCOPIC EXAMINATION OF THE BLOOD 75 

These granules are ordinarily very abundant; but in disease they 
may diminish in number until very few are left, and in some cases 
they may indeed be absent. Ewing 1 has called especial attention to 
the decrease in the number of the granules in the acute leukocytosis. 
I have observed total absence of granules in a case of trichinosis at a 
time when marked eosinophilia existed. Kast mentions an instance 
of general carcinomatosis with a leukocytosis of 120,000 in which 
1.68 per cent, of the cells contained no granules. Hirschfeld de- 
scribes the same occurrence in connection with growths involving the 
bone-marrow, and others have noted it in myeloid leukemia, where 
toward the end, in chronic cases, it is a fairly common phenomenon. 

Associated with the diminution in the number of the granules there 
are frequently also degenerative changes affecting the nuclei. These 
may be of the type of karyolysis with swelling and loss of chromatin, 
or of karyorrhexis with hyperchromatosis and fragmentation of the 
nucleus. The former is the more usual in the acute leukocytoses, 
while the latter is seen especially in leukemia. In cases of the myeloid 
variety it is quite common to note complete fragmentation of the 
nucleus into six to ten segments. This phenomenon was first observed 
by Ehrlich in a case of hemorrhagic smallpox, and is of common occur- 
rence in fresh exudates. Cell degeneration associated with loss of 
chromatin and swelling, while it no doubt occurs to a greater degree 
in disease, may also be observed under normal conditions. In every 
dried and stained specimen a certain number of such cells will be found 
in which the nucleus appears as a much swollen and but faintly stain- 
ing shadow, the Kernschatten of the Germans, sometimes surrounded 
by some of the granules, which appear scattered as though the cell 
had been burst asunder by force; at other times the Kernschatten 
alone remains and nothing is seen of the body of the cell. 

I have stated that the loss of granules on the part of these cells 
may go on to a point where they are absent altogether. It may 
happen, however, that the granules are only apparently absent, and 
merely do not react as usual with ordinary dyes. A proper explana- 
tion of this peculiar behavior cannot be given, but every worker 
in blood is no doubt familiar with the phenomenon. Sometimes a 
change in the mode of fixation will cause the granulation to appear; 
at other times it may be demonstrated by the aid of some other dye. 

Vacuolization of the polynuclear leukocytes is very much less 
common than in the case of the mononuclear elements. 

While the neutrophilic leukocytes as a general rule are large cells, 
unusually small specimens are seen in the blood of myeloid leukemia. 
These dwarf forms must not be mistaken for the small cells which one 
may find in any specimen of blood where it is thick and where the pro- 
cess of drying has occurred slowly. In cells of this latter order the 

1 Clinical Pathology of the Blood, Lea Bros., 1st ed., p. 113 



76 THE BLOOD 

staining of the granules is also frequently deficient or they may not 
show at all. 

Neusser 1 some years ago called attention to the fact that with a 
certain modification of Ehrlich's triacid stain it is possible to demon- 
strate the presence of basophilic granules about the nucleus of some 
of the polynuclear leukocytes, as well as the mononuclear elements. 
He, as well as Kolisch, 2 regarded the presence of these perinuclear 
granules as characteristic of the so-called uric acid diathesis. As 
tubercular disease, moreover, is usually not seen in such cases, Neus- 
ser thought the presence of these granules in cases of phthisis to be 
a favorable symptom. Futcher, 3 on the other hand, was unable to 
confirm these observations, and my own investigations 4 are likewise 
opposed to Neusser's conclusions. I was able to demonstrate the gran- 
ules both in health and disease in almost every case, and was at one 
time even led to think that their absence was of more significance than 
their presence. A relation between their presence and the elimina- 
tion of uric acid or xanthin bases certainly does not exist. Within 
recent years the subject has received no further attention, especially 
since Ehrlich expressed the belief that the granules are artefacts. 
He states that they are only exceptionally seen when solutions of 
chemically pure crystalline dyes are used, from the Actiengesellschaft 
fur Anilinfarbstoife in Berlin. 

The polynuclear neutrophilic leukocytes are derived from corre- 
sponding mononuclaer forms — the neutrophilic myelocytes — which 
are normally found only in the bone-marrow. They result from 
these directly and represent their adult form. 

Arneth 5 divides the polynuclear neutrophiles into five classes accord- 
ing to the number of nuclear lobes. Under normal conditions the 
percentage numbers of the different varieties remain fairly constant 
for one and the same individual, but vary somewhat in different people. 
The first class is represented by mononuclear forms and is subdivided 
into (a) mononuclear forms, corresponding to and identical with Ehr- 
lich's myelocytes (see below); (b) forms with but slightly indented 
nuclei, the invagination not extending to a greater depth than the 
middle of the nucleus (the metamyelocytes); (c) cells in which the 
invagination extends farther than in form (6), but in which no separa- 
tion into isolated loops or lobes has as yet occurred — the true poly- 
morphonuclear variety. The two first varieties are essentially only 
seen under abnormal conditions, although an occasional metamyelo- 
cyte may at times be encountered in health. Cells of type (c) are 
present to the extent of 4 to 9 per cent. The second class com- 
prises cells with two distinct nuclear fragments, which may appear 

1 Wien. klin. Woch., 1894, p. 71. 2 Ibid., 1893, p. 797. 

3 Johns Hopkins Hosp. Bull., May, 1897. 

4 Amer. Jour. Med. Sci., vol. cxvii, p. 139. 

5 Die neutrophilen weissen Blutkorperchen. G. Fischer, Jena, 1904. 



MICROSCOPIC EXAMINATION OF THE BLOOD 77 

either as two loops or two lobes. They constitute from 21 to 47 
per cent.; the number, as already stated, varies somewhat with the 
individual, but is quite constant for one and the same person. In 
this class the cells with two loops normally always exceed those 
with one loop and one lobe, while true bilobes are rare. The third 
class presents three nuclear divisions and can be subdivided into 
four groups in accordance with the number of loops or lobes (see 
p. 81). Cells with two lobes and one loop approximate those with 
two loops and one lobe, while cells with three loops or three lobes 
respectively are in the minority. Conjointly the groups of the third 
class represent 33 to 48 per cent. Their number thus about equals 
that of group two, but has a tendency to be somewhat in advance. 
The fourth class is provided with four nuclear divisions with five 
subgroups and numbers 9 to 23 per cent. The fifth class finally 
comprises cells with five or more nuclear subdivisions and may be 
subdivided according to the same principle. Only 2 to 4 per cent, 
of the neutrophiles normally belong to this order. The various 
classes as just described represent different stages in the develop- 
ment of the neutrophilic cells, the myelocytes on the one hand being 
the youngest, and the polynuclear leukocytes with many lobes the 
oldest. 

The polynuclear neutrophiles are the most common leukocytes of 
the blood and normally constitute from 60 to 70 per cent, of the 
total number. In young children they are relatively less numerous 
excepting during the first twenty-four hours of life, when they may 
number 73 per cent. But they rapidly diminish, so that values of 
from 20 to 40 per cent, may be regarded as normal during the first 
year. Low values continue practically to the twelfth year, though 
the numbers gradually rise. From the twelfth to the fourteenth year 
60 per cent, may be regarded as an average; after that age the values 
given for the adult hold good. 

4. The Polynuclear Oxyphilic or Eosinophilic Leukocytes (Plate VI) . 
— In size and general appearance these cells resemble the polynuclear 
neutrophiles, and, like these, are capable of progressive locomotion. 
The granules — the ^-granulation of Ehrlich — however, are much 
larger and highly refractive, and possess a marked affinity for acid 
dyes, such as acid fuchsin and eosin. Hence the term oxyphilic 
or eosinophilic leukocytes. With neutral dyes or basic dyes they 
will not stain. The appearance of the individual granules varies 
somewhat in stained preparations. Some are round, others oval; 
some appear to stain throughout, others make the impression of 
little vesicles with a limiting membrane, which alone takes the dye, 
while the interior remains unstained. This bleb-like appearance 
of the granule is one of the most marked characteristics. Barker 1 

1 Johns Hopkins Hosp. Bull., 1894, p. 93. 



78 THE BLOOD 

has shown that the granules contain iron. They are insoluble in 
ether and cannot be stained with osmic acid. They are therefore 
not composed of fat. 

The protoplasm of the eosinophilic leukocytes is usually almost 
altogether hidden from view, owing to the dense packing of the 
granules; it is slightly basophilic. The nucleus is mostly bilobed, 
sometimes trilobed, and in stained specimens it is quite common 
to find the individual lobes unconnected by threads of chromatin; 
often the two lobes are situated at opposite poles. As a rule the 
nucleus is less markedly basophilic than that of the neutrophilic 
variety. A nucleolus is not seen. 

The same degenerative changes which have been described in con- 
nection with the polynuclear neutrophiles may also be observed in 
the eosinophils, and here, as there, one can at times note a material 
diminution in the number of the granules. I have never observed 
their entire absence, however, and it is noteworthy that in those 
cases of chronic leukemia in which the neutrophilic granulation may 
disappear the eosinophilic variety remains. 

While the common eosinophile is a large cell, unusually small eosino- 
philes are frequently seen in the blood of myeloid leukemia. These 
should not be confounded with the small forms which may be seen in 
the thicker portions of almost any normal specimen , and which latter 
owe their small size to a gradual contraction during the process of 
drying. 

Under normal conditions the percentage of the eosinophiles varies 
between 1 and 4. 

While repeated attempts have been made to connect the eosino- 
philic leukocytes of the blood cytogenetically with the neutrophilic 
variety, there is no satisfactory evidence to support this view. On 
the contrary, there are strong reasons for believing with Ehrlich 
that, analogous to the neutrophilic variety, the polynuclear eosino- 
philes are normally formed in the bone-marrow, and here only from 
mononuclear eosinophilic cells — the eosinophilic myelocytes. 

5. The Mast-cells (Polynuclear Basophilic Leukocytes) (Plate VI). 
— The mast-cells which are normally found in the blood are approxi- 
mately of the same size as the polynuclear neutrophiles and eosino- 
philes. In myeloid leukemia, however, in which they are espe- 
cially numerous, the size is more variable ; on the one hand, they may 
measure only 3.5 /j. in diameter, while on the other they may attain 
a dimension of 22 f±. The nucleus is polymorphous; but the ten- 
dency to form individual lobes is far less marked than in the corre- 
sponding eosinophilic and neutrophilic elements. Quite commonly 
it is leaf-like and flat in appearance. Its affinity for basic dyes is 
quite feeble, so that it is often difficult in stained preparations to 
make out the boundary line between nucleus and protoplasm. It is 
almost always excentrically located and usually has a fairly uniform 



MICROSCOPIC EXAMINATION OF THE BLOOD 79 

diameter of 4 t u. In the smaller specimens the nucleus occupies 
almost the entire cell. 

Embedded in the protoplasm lie granules of variable size — the 
y granulation of Ehrlich — some of which are fully as large as or even 
larger than the eosinophilic granules, while others are much finer. 
They are characterized by their affinity for basic dyes and the fact 
that with certain ones they stain metachromatically, viz., in a color 
which is different from that of the dye itself, which latter must be 
simple and not compound. Tissue elements which will stain in this 
manner are spoken of as chromotropic elements. Only a limited 
number of dyes have metachromatic properties. The most notable 
ones are the violet basic dyes hexamethyl violet, cresyl violet, thionin, 
neutral violet, and amethyst violet; further, the blue dyes methylene 
azure, cresyl blue, and toluidin blue, and the red basic dyes pyronin, 
acridin red, neutral red, and saffranin. With the latter group the mast- 
cell granules are colored yellow, with most of the violet dyes red, and 
with cresyl-violet R (extra) almost a pure brown. Methyl green 
does not stain the mast-cell granules unless it is contaminated with 
methyl violet, and for this reason the granules remain colorless in 
specimens stained with Ehrlich's triacid stain. In specimens fixed 
by heat and stained with aqueous alum hematoxylin solution the 
^-granules are also not demonstrable. They have been dissolved; 
but there remains a well-defined spongioplasm, upon which the gran- 
ules were deposited. 

The mast-cell granules are absolutely basophilic, viz., they can 
only be stained with basic dyes, and retain the basic dye on subse- 
quent differentiation in acid media. They are capable, moreover, 
of taking up the basic dye from its acidified solutions, as in the case 
of Ehrlich's dahlia-acetic acid mixture. 

The granules of the common mast-cells of normal blood are re- 
sistant to water, while in myeloid leukemia cells are met with the 
granules of which dissolve with great readiness. Their chemical 
nature is still a matter of dispute, but there is a tendency to associ- 
ate the mast-cell with the formation of mucin. This presupposes 
the identity of the blood mast-cell with the common mast-cell of 
connective tissue. In the past this has been tacitly assumed, but 
Pappenheim more especially has called attention to the fact that 
the hematogenic mast-cell differs from the histogenic form, and 
that the two probably represent different species. Pappenheim 
inclines to the view, however, that the granulation of the hema- 
togenous mast-cells is not a true morphological granulation, but 
merely chemically altered lymphocytic spongioplasm, or a transport 
substance which has been taken up and metabolized. 

The number of mast-cells varies between 0.2 and 1.0 per cent. 
Ewing states that he constantly failed to find mast-cells in the better 
class of healthy subjects, while in hospital and dispensary cases with 



80 THE BLOOD 

minor ailments they appeared to be more numerous. My own obser- 
vations do not bear this out; in my experience they are invariably pres- 
ent in health irrespective of the general nutrition of the individual. 

The origin of the mast-cells of the blood has not been definitely 
ascertained. Ehrlich supposed that they originated from the con- 
nective-tissue cells as the result of hypernutrition, while Harris 
suggests that they may be derived from the large mononuclear 
leukocytes. According to Pappenheim, the mast-cell originates in 
the bone-marrow from a granular mononuclear type which corre- 
sponds to the eosinophilic and neutrophilic myelocytes. 

6. The Myelocytes. — The myelocytes are mononuclear granular 
cells, which are normally not found in the circulation, but are en- 
countered only in the bone-marrow. 

Generally speaking, they represent the juvenile forms of the poly- 
nuclear leukocytes of the blood, and we accordingly distinguish three 
varieties, viz., the neutrophilic, eosinophilic, and basophilic myelo- 
cytes. The two last-named varieties, according to our present ideas, 
age directly into the corresponding polynuclear forms — i. e., they 
become the common eosinophiles and the mast-cell. In the case of 
the neutrophilic variety it appears that two types exist, a smaller 
and a larger form, which Pappenheim 1 designates respectively as the 
trachychromatic and the amblychromatic form. These are onto- 
genetically derived, the first from the last, but only the trachychro- 
matic variety ages into the common polynuclear neutrophile of the 
circulating blood. The nucleus of the amblychromatic form as it 
matures likewise becomes polymorphous, but normally the cell 
remains an inhabitant of the bone-marrow even then. 

As regards the origin of the myelocytes, I incline toward Pappen- 
heim's view, according to which all three varieties result from the 
large lymphocytes through a process of heteroplastic differentiation. 

(a) The Neutrophilic Myelocytes. — These, as I have stated, 
are of two kinds. The one type, the amblychromatic myelocyte of 
Pappenheim, is a large cell provided with a relatively large, cen- 
trally located, round nucleus which stains but feebly with basic dyes. 
This is surrounded by a comparatively narrow zone of basophilic 
protoplasm which contains very fine neutrophilic granules. As 
the cell matures the nucleus becomes more or less invaginated and 
ultimately distinctly polymorphous. The protoplasm at the same 
time becomes relatively more abundant. Pappenheim speaks of 
this type as the heteroplastic promyelocyte. Such cells differ mark- 
edly in size from the common polynuclear elements which result 
from the second type of myelocyte. 

The second type, viz., the trachychromatic myelocyte, is a smaller 
cell, which is essentially characterized by the fact that its nucleus 

1 Virchow's Archiv, vols, clix and clx. 



MICROSCOPIC EXAMINATION OF THE BLOOD 



81 



stains quite intensely with basic dyes. The protoplasm is faintly 
oxyphilic and the granulation rather coarser than in the amblychro- 
matic variety. As this cell matures the protoplasm becomes rela- 
tively more abundant and the nucleus distinctly polymorphous; it 
then constitutes the common neutrophile of the circulating blood. 
Between these two extremes there are transition forms, in which the 
nucleus is still single, but already shows a marked tendency toward 
polymorphism. These cells do not occur in normal blood. They have 
been described especially by Arneth. Pappenheim terms them 
metamyelocytes ovproleukocytes (see Fig. 16). 



Ia 



16 



ii 



in 



IV 



W(£ 










Fig. 16.— Karyolobism and polynucleosis of neutrophilic leukocytes. 



Neutrophilic myelocytes undergoing mitosis are sometimes seen 
in the circulating blood in cases of myeloid leukemia; on the whole, 
however, they are rare, and it is more common to meet with cells 
in which the division of the nucleus has already taken place (Plate VI). 

Miiller and Jolly have shown that the neutrophilic myelocytes of 
the circulating blood are capable of active locomotion. 

(b) The Eosinophilic Myelocytes. — In the more mature forms 
the color of the eosinophilic granulation on staining with eosin- 
methylene-blue mixtures is a pure eosin red. The younger forms, 
6 



82 THE BLOOD 

however, present a purplish-violet color, and some granules may 
indeed be a pure blue (Plate VI). This appearance is owing to 
the fact that the young eosinophilic granule is physically cyanophilic 
and chemically amphophilic, whereas the mature granule is physically 
erythrophilic, but chemically absolutely oxyphilic. This is well 
shown by staining such young cells with a mixture in which the 
basic dye is of a light color and the acid component dark, such as 
vesuvin on the one hand and water blue on the other. The mature 
eosinophilic granules will then take on the blue color of the water 
blue, while the young granules which stained blue with the eosin- 
methylene-blue mixture, and which we might accordingly have 
regarded as basophilic, are now likewise colored by the acid blue 
instead of the basic vesuvin, thus showing that they are in reality 
not basophilic, but amphophilic-cyanophilic 

The protoplasm of the eosinophilic myelocytes is basophilic. 

The size of the cells is quite variable; some are considerably larger 
than the corresponding poly nuclear form, while others are much 
smaller. The cyanophilic cells are, generally speaking, the largest. 

According to the observations of Muller and Jolly the eosino- 
philic myelocytes are capable of progressive locomotion. 

(c) The Basophilic Myelocytes, like the eosinophilic and neu- 
trophilic varieties, may be of variable size and are provided with a 
large centrically located nucleus, which is often distinguished only 
with difficulty from the surrounding protoplasm. 

7. Irritation Form (Stimulation Forms, or Phlogocytes). — 
These are mononuclear non-granular cells, the protoplasm of which 
is stained a rich brown by the triacid mixture. The nucleus is round, 
eccentrically located, and colored a bluish green. Oftentimes it 
shows a distinct wheel-spoke structure. According to Turk, who 
first described these cells, they are met with under the same conditions 
as the myelocytes. Pappenheim regards them as plasma cells and as 
largely derived from histogenic lymphocytes as the result of a retro- 
gressive degeneration, and characterized by hypertrophy of the 
cytoreticulum, increase of chromatin and chromatokinesis of the 
nucleus with coincident appearance of a markedly chromatophilic sub- 
stance of exogenic origin. As intermediary cells Pappenheim regards 
lymphocytes without chromophilic protoplasm, but with radiary 
nuclei. It is thus essentially a pathological product. The cells 
have a spongioplastic cytoreticulum and vacuoles. They may attain 
a size of 30 /^. These cells, in my experience, are most frequently 
met with in the blood of children, where their number may attain 5 
per cent, of all leukocytes. Wrench and Bryant 1 found this propor- 
tion in a girl of 10, in which, possibly as the result of gas poisoning, 
a severe anemia had developed. 

1 Guy's Hosp, Repts., 1905, vol. lix, p. 333. 



MICROSCOPIC EXAMINATION OF THE BLOOD 83 

According to Pappenheim the occurrence of plasma cells in the 
blood is indicative of a chronic inflammatory process, either of the 
connective tissue or of the hemopoietic apparatus (tuberculosis, 
Hodgkin's disease, myeloma, etc.). I have found them relatively 
numerous in inflammatory conditions of the abdominal viscera (peri- 
tonitis, appendicitis, typhoid fever), and occasionally in measles. 

The term irritation or stimulation forms indicates that the cells 
are found in connection with infectious, toxic, viz., inflammatory 
"irritation." 

Iodophilia. — On staining blood smears of normal individuals with 
iodine (see p. 137) the protoplasm of the leukocytes is colored a 
bright yellow, while the nucleus is somewhat refractory and takes on 
a lighter tint. Under certain pathological conditions this staining 
quality is modified; cells are then seen in which reddish-brown 
granules appear in the protoplasm or it may occur that this presents 
a diffuse brownish color throughout. This intracellular reaction 
affects the polynuclear neutrophiles almost exclusively; the mono- 
nuclear elements may, however, also react, in which case one com- 
monly sees large, pale-brown granules arranged about the nucleus in 
a single row. In eosinophiles the reaction does not occur. The 
extent to which the leukocytes are involved is quite variable; in 
some cases a few cells only are affected, while in others one is scarcely 
able to find a normal cell in an entire preparation. 

An extracellular reaction also occurs, but is of little clinical in- 
terest, as it is not infrequent even in health; it occurs in small, round- 
ish or oval masses, which are possibly true plaques, but which may 
also be small bits of protoplasm derived from leukocytes. 

As to the nature of the substance which reacts with the iodine in 
the manner indicated, there is no uniformity of opinion. Ehrlich 
regards it as glycogen, and assumes that this is present normally in 
every cell in the form of a colorless compound, from which the free 
glycogen is under certain conditions split off, and can then be demon- 
strated as such. Czerny, on the other hand, looks upon the iodo- 
philic substance as an antecedent of amyloid, while Goldberger 
and Weiss view it as peptone. Kaminer has shown that normal 
bone-marrow does not contain iodophilic leukocytes, but that they 
may here be found when they are present also in the blood. He 
concludes that the reaction is a degenerative phenomenon and not an 
evidence of regeneration. 

The occurrence of the reaction in disease has been studied especially 
by Gabritschewsky, Czerny, Livierato, Kaminer, Cabot, and Locke. 
From these investigations it appears that septic conditions of all 
kinds may furnish a positive reaction, but that active suppuration 
may also occur without iodophilia (Reich, Kiittner). Locke's list 
of diseases of this order includes general septicemia, abscesses (ex- 
cepting in the earliest stages), appendicitis accompanied by abscess 



84 THE BLOOD 

formation, general peritonitis, empyema, pneumonia, pyonephrosis, 
salpingitis with severe inflammation or abscess formation, tonsillitis, 
gonorrheal arthritis (in contra-indication to other forms), and acute 
intestinal obstruction where the bowel has become gangrenous. 
Locke concludes that no septic condition of any severity can exist 
without a positive reaction. In puerperal sepsis also it is said to be 
constant (Kaminer). In pneumonia with frank resolution it dis- 
appears in from twenty-four to forty-eight hours following crisis. 
In typhoid fever a positive reaction is not commonly obtained before 
the end of the second week, and it may indeed remain absent through- 
out the course of the disease. In the differential diagnosis between 
a serous and a purulent pleuritic effusion the absence of the reaction 
points to the former condition. Cerebral abscess may show the re- 
action, while in brain tumor it is absent (Gulland). In diphtheria 
it is only seen when there is much inflammation; it is never intense 
(Gulland). 

In contradistinction to chlorosis, pseudoleukemia, and the common 
forms of secondary anemia of moderate intensity, iodophilic leuko- 
cytes are found only in the severer forms of anemia, such as perni- 
cious anemia, leukemia (notably in acute cases), and the severe forms 
of secondary anemia. 

In animals the reaction can be produced artificially by infection 
with the streptococcus, the staphylococcus, the Bacillus pyocyaneus, 
Loffler's bacillus, the anthrax bacillus, that of Friedlander, the Bacillus 
coli communis, or the typhoid bacillus; as also by means of ricin, 
abrin, and the diphtheria toxin. Following the injection of oil of tur- 
pentine, croton oil, mustard oil, and silver nitrate, the reaction may 
occur even though bacterial infection has been avoided. In man it 
is also said to occur following narcosis. 

Literature. — Ehrlich, Zeit. f. klin. Med., 1882, vol. vi. Gabritschewsky, 
Arch. f. exp. Path. u. Pharmak., 1891, vol. xxviii. Czerny, ibid., 1893, vol. 
xxxi. Goldberger u. Weiss, Wien. klin. Woch., 1897. Hofbauer, Centralbl. f. 
inn. Med., 1899. Livierato, Deutsch. Arch. f. klin. Med., 1894, vol. liii. Kam- 
iner, Berl. klin. Woch., 1899, p. 119; and Deutsch. med. Woch., 1899, p. 206. 
Cabot and Locke, Jour. Med. Research, 1902, vol. vii. Locke, Boston Med. and 
Surg. Jour., 1902, p. 289. Reich, Beit. klin. Chir., xlii, 2. Kiittner, Arch. f. 
klin. Chir., lxxiii, 2; and Centralbl. f. Chir., 1904, No. 27, Beil., pp. 3-5. 

Leukocytosis. — While the number of red corpuscles is normally 
fairly constant, that of the leukocytes is subject to not inconsiderable 
variation. It is influenced by the age and sex of the individual, the 
process of digestion, menstruation, pregnancy, the bloodvessel from 
which the specimen is taken, etc. Generally speaking the number 
of the leukocytes varies between 3000 and 10,000, the exact num- 
ber, ceteris paribus, depending upon the state of nutrition of the 
individual. In ill-nourished persons low values are the rule, while 
maximum numbers are generally associated with a state of excep- 



MICROSCOPIC EXAMINATION OF THE BLOOD 85 

tional vigor and good nutrition. These extreme figures, however, 
are uncommon, and as a general rule a count of 10,000 may be re- 
garded as abnormal; 5000 to 6000 are the most common values 
which one finds if the examination is made with the individual in a 
fasting condition. During the process of digestion the figures are 
higher (see below). 

An increase in the number of leukocytes is met with under the 
most diverse conditions, both in health and disease. When transi- 
tory, it is designated as leukocytosis. But it would be better to restrict 
this term to indicate the number of the leukocytes in a general way, 
and to speak of an increase as hyperleukocytosis, and of a decrease as 
hypoleukocytosis. 

It will be convenient to consider the subject of leukocytosis under 
the following headings: 

la. Polynuclear neutrophilic hyperleukocytosis. 

16. Polynuclear neutrophilic hypoleukocytosis. 

2a. Polynuclear eosinophilic hyperleukocytosis. 

26. Polynuclear eosinophilic hypoleukocytosis. 

3a. Mast-cell hyperleukocytosis. 

36. Mast-cell hypoleukocytosis. 

4a. Large mononuclear hyperleukocytosis. 

46. Large mononuclear hypoleukocytosis. 

5a. Lymphocytosis. 

56. Lymphopenia. 

The term myelemia, or myelocytosis, may be used to designate the 
appearance of myelocytes in the circulating blood, and in conformity 
with the three recognized forms we may speak of a neutrophilic, an 
eosinophilic, and a basophilic or mast-cell myelocytosis. 

Until quite recently the general tendency in clinical laboratories has 
been to lay especial stress upon the absolute leukocyte count and to leave 
the relative values of the different forms out of sight. This should not 
be, and I cannot insist too strongly upon the importance of the relative 
count, which in many respects is far greater than a knowledge of the 
total number. For this reason also I have chosen the consideration of 
the subject of hyperleukocytosis on the basis of the classification just 
outlined. 

Polynuclear Neutrophilic Hyperleukocytosis. — This is the most 
common form of hyperleukocytosis, and, as the term indicates, 
principally affects the polynuclear neutrophiles. Exceptionally it 
may be associated with a polynuclear eosinophilia, as well as with a 
lymphocytosis: but as a general rule both eosinophiles and lympho- 
cytes are diminished. This diminution is often not only relative, 
but absolute as well. In very marked cases of hyperleukocytosis of 
this type it is not uncommon to meet with a few myelocytes which 
are then also of the neutrophilic variety; this is especially the case 
in children in whom the bone-marrow reacts more readily to stimu- 



86 THE BLOOD 

lation. Eosinophilic myelocytes, on the other hand, are but rarely 
seen. 

Clinically we must distinguish between an increase of the poly- 
nuclear neutrophiles which may occur in health and the common 
hyperleukocytosis which is observed in disease. We may accord- 
ingly speak of a physiological and a pathological form. 

Physiological Hyperleukocytosis. — As physiological increase in the 
number of the leukocytes is notably observed at birth, during the 
process of digestion, in pregnancy, in association with severe mus- 
cular exercise, following the use of cold baths, etc. 

Leukocytosis of the Newborn. — According to the experience of 
most observers, the number of leukocytes at birth varies between 
10,000 and 23,000, of which over 70 per cent, are polynuclear neu- 
trophiles. The number then falls and at the same time the lympho- 
cytes increase. The curves of the two varieties cross between the 
sixth and the ninth day, and by the twelfth the lymphocytes are in 
excess. From the end of the first month to the fourteenth year there 
occurs a gradual increase of the neutrophiles and a decrease of the 
mononuclear elements. During the first year the total number of 
the leukocytes varies between 10,900 and 12,900; 9000 may be 
regarded as an average value from the first to the sixth year, and 7900 
from then until the fifteenth year. 

Digestive Leukocytosis. — The increase in the number of the leuko- 
cytes which is observed during the process of digestion affects both 
the polynuclear elements and the lymphocytes, though especially the 
latter. The eosinophiles are relatively at least diminished. The 
total increase rarely exceeds 3500 in normal adults, while in young 
children it may be much more marked. Schiff * cites an instance in 
which 19,500 leukocytes were counted one hour after birth, 27,625 
after the first meal, and 36,000 after the fourth meal. It is especially 
pronounced after a preliminary period of fasting and following a meal 
rich in proteids. The maximum increase is usually observed between 
the third and fourth hour. 

In cases in which a hyperleukocytosis exists from other causes, as 
in pregnancy, in inflammatory diseases, etc., digestive hyperleuko- 
cytosis does not occur. Lobenstine 2 in analyzing 20 cases of pregnancy 
in this direction found digestive leukocytosis in 13, no change in 1 and 
an actual decrease in 6. Apparently, however, he only made his 
examinations following the ordinary midday meal. In a few isolated 
instances it has also been found absent in apparently normal individ- 
uals without assignable cause. Under pathological conditions its 
absence is not uncommon, even though hyperleukocytosis referable 
to other factors may not exist. This is notably the case in carcinoma 

1 Zeit. f. Heilk., vol. xi, p. 30, and 1890, p. 1. 

2 Amer. Jour. Med. Sci., August, 1904. 



MICROSCOPIC EXAMINATION OF THE BLOOD 87 

of the stomach, and it was once thought that the absence of digestive 
hyperleukocytosis in doubtful cases could be interpreted as evidence 
in favor of its existence. 1 Generally speaking, this is true even now, 
and we may say that in about 90 per cent, of all cases of carcinoma of 
the stomach digestive hyperleukocytosis does not occur. The symp- 
tom, however, is not pathognomonic, as a number of instances of 
carcinoma have been reported in which there was a distinct increase, 
and as digestive leukocytosis may also be absent in other conditions. 
In anemic individuals, from whatever cause, especially large amounts 
of proteids are sometimes necessary to elicit an increase of the leuko- 
cytes (Miiller 2 ) and in some cases a subnormal number may even be 
encountered (Rieder 3 ). 

To study digestive hyperleukocytosis, it is well to proceed as 
follows : 

(a) The first blood count should be made after the patient has 
fasted for about seventeen hours. 

(b) After this period he receives a test meal consisting of from 
200 to 1000 c.c. of milk and one or two eggs, the amount varying 
with the condition of the patient. 

(c) Further blood counts are made one, two, three, and four hours 
later. 

(d) The existence of a digestive hyperleukocytosis should only be 
regarded as proved if an increase of at least 1500 cells occurs, pro- 
viding that maximal amounts of food have been taken. If smaller 
amounts have been given, an increase of 1000 cells is sufficient to 
establish its existence, provided that the same result is observed on 
repeated examination. 

Leukocytosis of Pregnancy and Parturition. — The hyperleukocy- 
tosis which is observed in pregnancy is particularly marked during 
the last five months, and appears to occur quite constantly in piimi- 
parse, while in multiparas exceptions are common. In an analysis 
of 55 cases Hubbard and White 4 obtained positive results in 44 — 
i. e., in 80 per cent. — most marked and constant in young primiparse. 
Rieder in an analysis of 31 cases noted a hyperleukocytosis in 20, 
all the negative cases being multipara?. In a series of 17 multiparas 
an increased number of leukocytes was noted in only 7. In Rieder's 
series the number of leukocytes varied between 10,000 and 16,000, 
with an average of 13,000. This represents the usual increase, but 
at times much larger numbers may be observed; Cabot thus reports 
3 cases with a leukocytosis of from 25,000 to 37,000 

Lobenstine gives the following figures as the result of an analysis 
of 50 cases in the ninth month: 

1 Schneyer, "Das Verhalten d. Verdauungsleukocytose b. ulcus rotundum u. 
carcinoma ventriculi," Zeit. f. klin. Med., vol. xxvii, p. 219. 

2 Zeit. f. Heilk., 1890, p 213. 

3 Beit. z. Kenntniss d. Leukocytose, 1892. 

4 Jour, of Exper. Med., 1898, p. 639. 



88 THE BLOOD 

Average count 10,600 

Highest count 18,000 

Lowest count 5,400 

Average in primiparse . . . 9,346 

Average in multipara . 11,854 

Absence of leukocytes in 7 cases. 

During actual labor there is an increase of the leukocytes over and 
above the numbers previously observed in pregnancy; 30,000 cells 
may then be noted. 

Lobenstine's figures in his series of 50 normal cases on the third 
day of the puerperal period are the following: 

Average count 12,400 

Highest count 20,400 

Lowest count 5,600 

Average in primiparse 13,200 

Average in multiparas 11,600 

No leukocytosis in 8 cases. 

The highest numbers are met with in severe and protracted cases, 
especially after rupture of the waters. This form of hyperleukocytosis 
subsides after the expulsion of the child, and at the end of the first 
or second week normal values are again reached, though the gradual 
decline may be interrupted by a temporary increase now and then, 
referable to various minor disturbances during the puerperal state. 
In many cases normal values are reached much earlier, and by the 
third day, as a rule, the number is as low as it was before labor. 

As in the case of the digestive leukocytosis, the hyperleukocytosis 
of pregnancy and the puerperal state is brought about by an increase 
both of the polynuclear neutrophiles and the lymphocytes, while the 
eosinophiles remain passive. 

Leukocytosis following Baths, Muscular Exercise, etc. — The in- 
crease of the leukocytes following cold baths may, according to 
Thayer, 1 amount to nearly 300 per cent. In 20 cases of typhoid 
fever he found 7724 leukocytes on an average before and 13,170 
after the usual Brand bath. In his own person, while in health, the 
leukocytes on one occasion numbered 3250 before the bath, while 
twenty minutes later they had increased to 12,500. Such an increase 
is, however, only observed after a bath of moderate duration, while 
a prolonged cold bath diminishes the number. Hot baths have 
exactly the opposite effect, viz., those of short duration produce a 
decrease, those of long duration an increase. 

Violent muscular exercise, as well as massage, produces a tempo- 
rary hyperleukocytosis. 

Pathological Hyperleukocytosis. 1. The Hyperleukocytosis of the 
Acute Infections. — In the acute infectious diseases hyperleukocytosis 
referable to an increase of the polynuclear neutrophiles is the rule. It 

1 Johns Hopkins Hosp. Bull., April, 1893. 



MICROSCOPIC EXAMINATION OF THE BLOOD 89 

is seen in pneumonia, erysipelas, diphtheria, scarlatina, the various 
pyogenic infections in the narrower sense of the term, in parotitis, 
acute articular rheumatism, epidemic cerebrospinal meningitis, etc. 
Typhoid fever and measles represent notable exceptions if we disregard 
the very earliest stage in the development of the disease, when an 
acute hyperleukocytosis may also be observed (see p. 97). 

Generally speaking, the increase in the number of the leukocytes 
in the acute infectious diseases is directly proportionate to the inten- 
sity of the infection and the power of resistance on the part of the 
individual. Where this is particularly feeble or the virulence of the 
infection is especially intense, an absolute increase of the total number 
of the leukocytes may not take place, although a relative increase of 
the polynuclear neutrophilic elements will probably always be observed. 
A recognition of this fact is of importance and serves to illustrate the 
special value of the differential count. 

In pneumonia the increase in the number of the leukocytes is 
usually marked. On an average it amounts to about 24,000 cells 
above the normal (Cabot). In rare cases a leukocytosis of 100,000 
and over has been observed. The hyperleukocytosis sets in quite 
early — within a few hours following the initial chill — and persists until 
the time of the crisis, when it rapidly disappears; the decrease may 
indeed precede the critical fall of the temperature. When the disease 
terminates by lysis the return to the normal is more gradual. A 
pseudocrisis is not accompanied by a fall in the number of the leu- 
kocytes. When resolution is delayed or complications occur, the 
hyperleukocytosis persists. 

Absence of hyperleukocytosis, excepting in very mild cases, will 
usually warrant a fatal prognosis; exceptions, however, occur, and it 
is well in any case to base prognostic conclusions not upon a single 
count, but upon the result of repeated examinations, as it is not 
uncommon to meet with considerable fluctuations in the course 
of the disease. Sears and Larrabee 1 found the mortality much 
greater when the leukocytes numbered less than 10,000 than when 
they were more numerous; and, according to Loper, a progressive 
increase of the neutrophiles beyond 90 to 95 per cent, may be regarded 
as an unfavorable symptom irrespective of their total number. Asso- 
ciated with the increase of the polynuclear neutrophiles in pneumonia 
there is a relative diminution of the lymphocytes. The eosinophil es 
are greatly diminished; they may indeed be absent. Their return 
may occur before the beginning of the crisis and may be viewed as 
a favorable symptom. 

In bronchopneumonia the total increase of the leukocytes is not so 
great as in the acute croupous form. 

In erysipelas, as in pneumonia, the hyperleukocytosis is generally 

1 Med. and Surg. Rep. of the Boston City Hosp., 1901, 12th series, Dee. 1st. 



90 THE BLOOD 

proportionate to the intensity of the morbid process and also termi- 
nates by crisis. The increase of the leukocytes beyond normal may 
amount to 15,000; in many cases, however, the total number does 
not rise much beyond the upper limit of the normal. At the height 
of the disease the eosinophiles are much diminished or absent. 

In diphtheria a well-marked increase is the rule. Generally the 
count does not exceed 25,000 to 30,000, but in fatal cases it is com- 
mon to meet with larger numbers. Ewing 1 speaks of one case with 
lymphocytosis in which the count was 72,000, and cites a peculiar 
instance reported by Felsenthal 2 marked by hemorrhagic eruption in 
which 148,000 were counted. As Ewing suggests, this was probably 
an agonal hyperleukocytosis. As a rule, from 25,000 to 50,000 cells 
are met with in fatal cases. In children the general increase of the 
leukocytes is frequently associated with a relative lymphocytosis. 
The eosinophiles are diminished in number and may indeed be 
absent. It is interesting to note that excepting a temporary dimi- 
nution immediately following the injection the leukocytosis is in no 
wise influenced by the antitoxin treatment. Besredka, 3 however, 
states that the grade of the polynuclear neutrophilic hyperleukocytosis 
after the administration of the serum indicates the prognosis. Thus, 
if one or two days after the injection the percentage of the neutro- 
philes is 60 or more, the prognosis is good; with a higher tempera- 
ture and 50 per cent, it is bad, and with a lower percentage the disease 
is fatal. Simon 4 finds that the occurrence of hyperleukocytosis and 
hyperpolynucleosis four hours after the injection is a favorable sign. 
The exanthem which occasionally follows the injection of antidiph- 
theritic serum is accompanied by a polynuclear neutrophilic hyper- 
leukocytosis. 

In tonsillitis there is an increase of the leukocytes of approximately 
the same intensity as in diphtheria, with a similar diminution in the 
number of the eosinophiles. 

In septic conditions, in general, hyperleukocytosis is of constant 
occurrence at some stage of the disease, unless the infection is very 
mild or very severe. Even in those cases in which the absolute increase 
of the leukocytes is not marked, or, as in certain very virulent cases 
absent altogether, the neutrophils are relatively increased and the 
eosinophiles coincidently very much diminished or absent altogether. 
This association I have termed the septic factor and I cannot insist 
too strongly upon its value in the diagnosis of acute infections with 
the group of pyogenic organisms. 

Especially important is the study of the leukocytosis in appendicitis. 
According to Curschmann's initial studies in this direction a leukocy- 

1 The Blood, loc. cit. 

2 Arch. f. Kinderheilk., vol. xv, p. 78. 

3 Annal de l'lnst. Pasteur, 1898, vol. xii, 5, p. 305. 

4 Journ. de physiol. and pathol. gen., vol. v, p. 887. 



MICROSCOPIC EXAMINATION OF THE BLOOD 91 

tosis of 22,000 is strongly suggestive of an existing abscess; if the 
count remains stationary at this point, or if it . increases but once 
to 25,000, suppuration may be regarded as established. These results 
have been largely confirmed by other investigators. Exceptions, 
however, occur and surgeons perhaps not unnaturally decline to be 
guided in their operative work by the results of the blood count only. 
Personally I value the absolute count very highly in the study of the 
progress of an appendicitis, but only in so far as an increase, and, above 
all, a progressive increase is concerned. Normal figures or a decreas- 
ing leukocytosis are very dubious factors and should be viewed with 
reserve. In my estimation the differential count is much more valu- 
able in the study of these cases and far less apt to mislead. A decreas- 
ing neutrophilic hyperleukocytosis with a return of the eosinophiles 
is a favorable symptom in all cases. A drop in the total count with 
a continuance of the relative polynucleosis, on the other hand, usually 
means added danger and only too often perforation. 

I here append extracts from Bloodgood's paper, 1 which gives a 
good idea of the usual findings in appendiceal and related abdominal 
affections, but regret that the absolute counts only have been con- 
sidered. 

Observed within forty-eight hours the number of white blood cells 
is in the majority of instances of great value in pointing to the extent 
of the inflammatory condition of and about the appendix. Cases 
of recurrent appendicitis or of appendicitis suffering from the first 
attack, first observed practically at the end of the attack when the 
clinical symptoms are subsiding, rarely show an increase in the 
white cells. In a few instances, first observed within forty-eight 
hours after the beginning of the attack, but when the symptoms 
are subsiding, there have been a few leukocyte counts of 15,000, 
which have fallen rapidly within a few hours to 10,000 and 7000. 
In the cases admitted within forty-eight hours with acute symptoms, 
if on account of the clinical picture operation has been delayed, a 
falling leukocytosis has always been observed. These patients have 
recovered, and at a later operation the appendix was found to be 
the seat of a diffuse inflammation, but there was no evidence of 
pus outside the appendix. In one case admitted sixteen hours after 
the beginning of the attack the leukocytes fell in ten hours from 
17,000 to 13,000, and in twenty-four hours to 11,000, associated with 
disappearance of the symptoms. With one exception, the highest 
first leukocyte count in this group has been 17,000, falling in a few 
hours to 12,000, 9000, or even lower. A patient admitted twenty 
hours after the beginning of the acute attack had a leukocytosis of 
22,000; the clinical symptoms, however, were not very marked. 
The patient was observed eight hours; during this period the leuko- 

1 Blood Examination as an Aid to Surgical Diagnosis, Amer. Med., 1901, p. 306 



92 THE BLOOD 

cytes fell to 16,000 and the local symptoms practically disappeared. 
Within the succeeding twenty-four hours the leukocytes were 11,000, 
then 8000, 7000, and 6000. Although this patient with a leukocytosis 
of 22,000 at the end of twenty hours, recovered, and there is every 
reason to believe that the inflammatory condition about the appendix 
subsided, nevertheless it is an exception to the general rule, and it 
would be safer, I believe, to operate in those cases of acute appendicitis 
observed within the first forty-eight hours with a leukocytosis of 
20,000. 

In acute diffuse appendicitis with operation and recovery the highest 
count observed was 25,000 thirty-six hours after the beginning of 
the attack. At operation in this case intense inflammation and a 
large amount of exudate were found about the appendix. 

In gangrenous appendicitis with operation and recovery the leuko- 
cytosis is higher (25,000 to 35,000) and rises more rapidly. As Blood- 
good says, the study of the leukocytosis is here of the greatest im- 
portance in the early recognition of a grave inflammatory condition 
of the appendix, which without doubt would lead to general peritonitis 
and death unless early operation be instituted. 

A very high leukocytosis within forty-eight hours after the begin- 
ning of the attack is suggestive, but not at all positive, of beginning 
peritonitis. The leukocyte count, however, does not seem to help in 
such cases with regard to prognosis. After the second day in cases in 
which the peritonitis has been present longer Bloodgood never has 
observed recovery with a low leukocyte count. If the leukocytosis 
remains still high at this period, however, the prognosis seems better 
for ultimate recovery after operation. 

In chronic suppuration the results are less decisive; there are cases 
indeed in which notwithstanding the existence of extensive intraperi- 
toneal accumulations of pus no increase of the leukocytes occurs. 

In intestinal obstruction an increase of the leukocytes associated 
even with very slight symptoms is of the highest importance in the 
early recognition of the lesion. Bloodgood states that in a large 
group of cases the leukocyte count may rise to 20,000 within twelve 
hours after the beginning of the obstruction. 1 Within the first twelve 
to twenty-four hours a few observations would demonstrate that if 
the leukocyte count rise above 25,000 or 30,000, the probabilities 
are that one will find gangrene of the obstructed loops or beginning 
peritonitis. If observed on the second or third day after the begin- 
ning of the symptoms, it is difficult to make a differential diagnosis 
with regard to gangrene or peritonitis. After the third day, in cases 
in which there is no gangrene and no peritonitis, or in which the 
auto-intoxication is not yet very grave, the leukocytes still remain 
high — 15,000 to 23,000 — according to the degree of obstruction: com- 

1 Cases have indeed been observed with an absolute count of 50,000, and more. 



MICROSCOPIC EXAMINATION OF THE BLOOD 93 

plete, higher; partial, lower. In the presence of gangrene peritonitis 
or grave auto-infection, the leukocytes begin to fall. If the patient 
is admitted after the third or fourth day, with a history of intestinal 
obstruction, and still has a high leukocyte count, the prognosis is 
good for operation. If the count is low, and especially if it is below 
10,000, the probabilities are that on operation extensive gangrenous 
peritonitis will be found; or the patient will be so depressed by auto- 
intoxication that reaction does not follow relief of the obstruction. 

In amebic liver abscess there is a comparatively low leukocytosis, 
viz., 10,000 to 17,000, rarely over 20,000. 

In eclampsia there is usually marked hyperleukocytosis, the degree 
coeteris paribus depending fairly closely upon the apparent toxicity of 
the case (16,000 to 20,000 in mild cases). With a good resistance the 
increase is especially marked (46,000 to 54,000). A sudden increase 
generally indicates an aggravation of the condition in an individual 
of good resistance (as high as 100,000). A low count in a highly toxic 
patient is of bad omen (19,000 dropping to 13,800 by the second day 
following delivery). A leukocytosis originally high that falls rapidly 
in a badly toxic patient is likewise a danger signal (100,000 to 45,200 
in one day) (Lobenstine). 

In the differential diagnosis between ruptured tubal pregnancy with 
associated severe internal hemorrhage and acute peritonitis a high 
leukocyte count speaks in favor of the first condition. In a slowly 
developing peritonitis, on the other hand, hyperleukocytosis may also 
be observed. With small hematoceles (referable to tubal pregnancy) 
the leukocytes may be normal. 

In pyosalpinx high leukocyte counts are almost always seen. 

In uterine carcinoma hyperleukocytosis is usually only seen when 
there is extensive ulceration. Myomas only lead to hyperleukocytosis 
as the result of extensive hemorrhages. In hydrosalpinx and salpingo- 
oophoritis the same is seen. In connection with ovarian cystoma the 
leukocyte values are usually normal; if, however, peritoneal irritation 
exists (as manifested by sensitiveness to pressure and ascites), a marked 
increase may occur. In such cases in contradistinction to pus cases 
the iodine reaction is negative. 

In scarlatina hyperleukocytosis is a constant feature of the disease. 1 
It usually begins two or three days before the appearance of the 
rash; sometimes even as early as the sixth day. The acme is reached 
on the second or third day; on the fourth medium values are found. 
Then the decrease usually begins, although this is sometimes delayed 
until the eighth or ninth day; normal values are not reached until 
the end of the second or the beginning of the third week. In light 
cases the leukocytosis amounts to from 10,000 to 20,000 cells, in cases 

1 Van der Berg, Arch. f. Kinderheilk., vol. xxv, p. 321. Mackie, Lancet, 
Aug. 24, 1901. Reckzeh, Zeit. f. klin. Med., 1902, vol. xliv, p. 201 (full literature). 



94 THE BLOOD 

of moderate severity 20,000 to 30,000 are average figures, while in 
fatal cases 40,000 are common values. The hyperleukocytosis is 
scarcely influenced by the height of the temperature, the angina, the 
rash, desquamation, or complications, excepting that in the latter 
case its duration is influenced by the nature of the pathological process. 
The hyperleukocytosis is due to a large increase of the polynuclear 
neutrophils, which may represent 94 per cent, of all leukocytes. 
The lymphocytes are proportionately diminished unless glandular 
complications occur, when they may reach maximum normal values. 
The eosinophiles in light and moderately severe cases are at first nor- 
mal or subnormal, they then gradually increase and reach maximum 
values (8 to 15 per cent.) in the second or third week, after which they 
return to normal. In severe cases they diminish to zero. 

In acute articular rheumatism the degree of hyperleukocytosis is 
proportionate to the severity of the attack. In McCraeV analysis 
of 83 cases the average count was 11,776; in 29 it was below 10,000. 
Taking the average of the remaining 54 cases we have 14,260. In 
17 the count was over 15,000 and in 4 over 20,000; the highest figure 
was 38,000. It is noteworthy that hyperleukocytosis was noted in all 
cases of complicating pericarditis in which a count was made, but 
that normal values were obtained in many cases of undoubted endo- 
carditis. In pericarditis 15,000 to 19,000 were average values; 
35,000 was the highest count noted. Generally speaking, when the 
number of leukocytes in acute articular rheumatism rises to 20,000 
or higher, pericarditis or pneumonia may be suspected (Turk, Ewing). 
When the total increase of the leukocytes is only slight, the percentage 
values are not especially disturbed, but with a marked hyperleuko- 
cytosis the polynuclear neutrophiles are materially increased. The 
eosinophiles are commonly absent in the early stages of the disease, 
while later they are always present in moderate numbers, and after 
defervescence they are usually increased. 

In tubercular disease hyperleukocytosis is observed only when 
secondary infection with pus organisms has taken place, while in 
pure cases the number remains normal. As the conditions for a 
secondary infection are more favorable in some parts of the body 
than in others, such as the lungs and kidneys, hyperleukocytosis is 
commonly present when these parts are involved. In the third 
stage of pulmonary tuberculosis there is usually a leukocytosis of 
from 15,000 to 20,000, which is referable to a well-marked increase 
of the polynuclear neutrophiles, while the eosinophiles are diminished. 
In the second stage, owing to a concentration of the blood no doubt, 
values ranging between 8000 and 10,000 are common, while in the 
first stage normal values are found. 2 In tubercular peritonitis the 

1 Jour. Amcr. Med. Assoc, 1903, vol. xl, p. 210. 

2 Appelbaum, loc. cit., p. 61. 



MICROSCOPIC EXAMINATION OF THE BLOOD 95 

leukocytosis is variable. In 36 cases of 46 analyzed by Shattuck 
the number was below 10,000; where it is higher pus may or may 
not be present. 

In the epidemic form of meningitis the count may range between 
32,000 and 34,000 early in the disease. Later, when the tem- 
perature rarely rises above normal the count may drop to from 
9000 to 10,000. In fatal cases the highest counts are usually seen 
(35,000 to 55,000), but it is to be noted that a high count does not 
necessarily imply a fatal ending. In the tubercular form hyper- 
leukocytosis is also observed in a large percentage of cases, but is not 
apt to exceed 24,000; in 60 per cent, of Koplik's cases it was under 
20,000. In many of these cases the hyperleukocytosis is due to a 
complicating terminal pneumonia. 

In serous non-tubercular pleurisy the leukocytes are not increased, 
while in the tubercular cases the number may rise to 15,000 to 20,000. 
This increase, however, is probably greatly dependent upon the extent 
of the primary lesion. 

In empyema there is marked hyperleukocytosis (22,000 to 29,000), 
which disappears after evacuation of the pus. 

In smallpox a hyperleukocytosis is observed only in the severer 
cases and when pustulation takes place. In the milder form no 
increase occurs. 

In Malta fever a marked increase of the polynuclear neutrophiles 
may occur just before the onset of the fever; later there is absence 
of hyperleukocytosis. In a case observed in the United States 
11,5.64 leukocytes were counted, all varieties being present in normal 
proportion. 1 

In bubonic plague a moderate increase of the leukocytes is the 
rule; a few instances have been reported in which over 100,000 
cells were counted, the increase being largely due to neutrophiles. 

In uncomplicated cases of typhoid fever, during the first few days, 
there may be a leukocytosis of 3000 to 5000 beyond the normal, 
referable to an increase of polynuclear neutrophiles. Subsequently 
they diminish and a relative lymphocytosis comes to the foreground 
(see especially p. 97). 

In tetanus a moderate neutrophilic increase occurs. 

In uncomplicated measles there is in the beginning a moderate 
relative increase of the polynuclear neutrophiles, 76 to 82 per 
cent.; but this is not associated with an absolute increase of the 
leukocytes, but with a decrease. Later there is a relative decrease 
of the neutrophiles to 50 to 60 per cent., while the absolute number 
is increased. 

According to Wilson and Chowning, a hyperleukocytosis of about 
12,000 is usual in cases of the so-called spotted fever of the Rocky 

1 Musser and Sailer, Phila. Med. Jour., 1898, p. 1408, and 1899, p 89. 



96 THE BLOOD 

Mountains. 1 But I have also seen the blood from several cases 
in which no increase existed. 

In some cases of enterogenous cyanosis the number of leukocytes 
may be increased to 20,000, but as a rule it varies between 4000 and 
10,000. The relative figures are scarcely affected. 

In dementia paralytica, throughout the disease, there is an increase 
of the polynuclear neutrophiles which reaches its height during the 
terminal stage, while the eosinophiles are for the most part materially 
diminished. Paralytic attacks are accompanied by a further increase 
of the neutrophiles. 2 

2. Anemic Hyperleukocytosis. — Hyperleukocytosis referable to an 
increase of the polynuclear neutrophiles is observed in various forms 
of acute and chronic secondary anemia. It is especially marked 
after hemorrhages, when the number of leukocytes may increase to 
30,000 and even more. Generally speaking, the degree of increase is 
proportionate to the amount of blood lost and the recuperative power 
of the individual. Rieder noted a leukocytosis of 15,000 after a 
pulmonary hemorrhage; 32,600 after a hemorrhage due to uterine 
cancer, and 26,500 after a hemorrhage referable to gastric ulcer. 

If we except the myeloid type of leukemia, in which an absolute 
increase of the polynuclear neutrophiles is associated with a relative 
decrease, hyperleukocytosis is not met with in uncomplicated cases 
of the primary anemias. 

3. Cachectic Hyperleukocytosis. — A cachectic hyperleukocytosis has 
been described in connection with malignant disease, phthisis, etc. 
Ewing states that in the majority of cases of tertiary syphilis, tuber- 
culosis, and nephritis, in a large proportion of carcinoma cases and in 
a rather smaller proportion of sarcomas the cachexia is unaccompanied 
by hyperleukocytosis unless there is distinct local inflammation, 
necrosis, or hemorrhage. He suggests that the existence of a marked 
hyperleukocytosis in the course of a cachexia should lead to a search 
for one of these complications. Kast has described a remarkable 
instance of universal carcinomatosis with bone-marrow involvement 
in which the total number of leukocytes rose to 120,000, with 94.49 per 
cent, of neutrophiles, although septic complications did not occur. 

4. Antemortem Hyperleukocytosis. — An agonal hyperleukocytosis 
in the old sense of the term is now no longer accepted. If during 
the agone hyperleukocytosis exists it is dependent directly upon the 
character of the morbid process, and not upon the agonal condition. 

5. Hyperleukocytosis referable to Drugs. — Hyperleukocytosis refer- 
able to an increase of the polynuclear neutrophiles has been observed 
in cases of poisoning with potassium chlorate, arsenious hydride, 
illuminating gas, and coal-tar derivatives, such as antifebrin, phen- 

1 Jour. Amer. Med. Assoc, July 19, 1902, p. 131. 

2 Diefendorf, Amer. Jour. Med Sei., December, 1903. 



MICROSCOPIC EXAMINATION OF THE BLOOD 97 

acetin, etc. It follows the administration of atropine, quinine, the 
salicylates, thyroid extract, tuberculin, and the infusion of salt solu- 
tion. It is noted after prolonged anesthesia with chloroform and ether, 
when an increase of 5000 to 10,000 cells is quite common. This 
increase occurs after from six to forty-eight hours following the opera- 
tion, and persists for only a few hours. A postoperative increase 
of 10,000 or more beyond the normal value of the individual, and sus- 
tained for more than a few hours, should be looked upon with sus- 
picion 1 unless the case was a septic one from the start, when it may 
persist for several days. 

6. Hyperleukocytosis of Thermic Fever. — In thermic fever a high 
leukocyte count is apparently the rule, but there is considerable 
irregularity in the time and duration of the rise. Lewis and Pack- 
ard 2 report that in some of their cases a leukocytosis of from 12,000 
to 13,000 was noted on admission. In most of the cases in which 
there was a primary rise this was followed by a fall and then a second 
increase in their number. 

Polynuclear Neutrophilic Hypoleukocytosis (Leukopenia). — 
A diminution in the total number of the leukocytes is observed in 
only a comparatively small number of diseases, and is practically 
always referable to a decrease in the number of the polynuclear 
neutrophiles. It is notably observed in typhoid fever, measles, 
influenza, in certain anemic conditions, etc. 

In typhoid fever 3 hypoleukocytosis is so constantly seen that we 
can formulate the general rule that whenever an increase in the num- 
ber of the leukocytes is observed in a case of suspected typhoid fever it 
is more than probable that some complication exists or that the diagnosis 
is wrong. Exceptions to this rule are rare. In the very earliest 
days of the disease, possibly owing to a concentration of the blood, 
the result of starvation and diarrhea, higher counts are sometimes 
observed, but as the disease progresses the number soon diminishes, 
and in the later stages of the disease is practically always markedly 
below the normal. Not uncommonly they are less than 2000, and 
in some instances the number may indeed fall below 1000. The 
qualitative changes are especially important and fairly character- 
istic of the different stages of the disease. At first, while the 
temperature is steadily rising there is a neutrophilic hyperleuko- 
cytosis of moderate degree; this is associated with a moderate de- 
crease of the lymphocytes, while the eosinophiles disappear. Then 
the neutrophiles diminish and the period of the hypoleukocytosis 
properly speaking commences. During this stage, viz., the stage of 

1 Da Costa and Kaltever, Amer. Med., 1901, p. 306. 

2 Trans. Assoc. Amer." Phys., 1902, p. 409. 

3 Nageli, Deutsch. Archiv, lxvii, parts iii and iv. Kolner, ibid., lx, p. 221. 
Thayer, Johns Hopkins Hosp Bull., 1901, vol. iii, p. 500; and Studies in Typhoid 
Fever, Johns Hopkins Press, 1901, p. 487. 

7 



98 THE BLOOD 

continued fever, the neutrophils usually number from 3000 to 4000, 
as compared with 5000 to 6000 during the second half of the first 
week. The lymphocytes are now also diminished, but tend to rise 
toward the end of this period; the eosinophils are absent. During 
the third stage (remission) the neutrophiles decrease still further — 
1500 to 2500 — while the lymphocytes increase and a few eosinophiles 
appear. In the fourth stage (defervescence) the neutrophiles reach 
their minimum, 900 in severe cases, while the lymphocytes are rela- 
tively much increased and the eosinophiles gradually return to normal. 
The reascent of the neutrophiles then occurs very slowly, while coin- 
cidently there is a lymphocytosis which is especially marked in children 
and continues far into convalescence. Normal values are some- 
times not reached until after a couple of months. 1 

In the event of a relapse occurring during an afebrile period there 
is a distinct neutrophilic hyperleukocytosis, the actual number depend- 
ing upon the preceding counts, to which from 3500 to 5000 neutro- 
philes are added ; at the same time the eosinophiles disappear. Should 
a relapse occur in the third stage of the disease, then the eosinophiles, 
which have just begun to reappear, disappear abruptly. 

Favorable indications in cases of typhoid fever are an increase of 
the eosinophiles at the height of the disease; reappearance of the 
eosinophiles, indicating arrival at the third or fourth stage; an 
increase of the lymphocytes, which appears to begin only at a time 
when the severest part of the intoxication is over; not too great a 
decrease of the neutrophiles in the absence of complications. Un- 
favorable indications are: a marked decrease of all leukocytes, and 
especially of the lymphocytes; absence of hyperleukocytosis and a 
further decrease of the neutrophiles in the event of complications, 
which per se would call forth a hyperleukocytosis. 

In the event of complications the total number of the leukocytes 
frequently does not exceed the upper limit of the normal; but in 
such cases a differential count will reveal a relative increase of the 
neutrophiles. 

In cases of perforation there is frequently an increase in the total 
number of the leukocytes, which may, however, be quite transitory 
and escape observation unless an early examination is made and 
previous counts are available; for later, when peritonitis is general, 
the leukocytes are usually found diminished. In some instances 
there is no increase at the onset. 

In one of Cabot's cases the count before operation was 8300, and 
immediately afterward 24,000. Finney reports a case with 6500 
before and 10,600 after. In one of Cushing's cases there was an 
early recognized hyperleukocytosis which appeared before any 

1 In my experience the increase of the mononuclear elements affects not only 
the small mononuclears, but also the large mononuclears. 



MICROSCOPIC EXAMINATION OF THE BLOOD 99 

sign of general peritonitis had developed; 8400 before and 16,000 
after. In this patient it was interesting to note that following the 
operation the leukocytes fell to 4000; but immediately following 
the development of obstruction, due to kinking of the bowel, the 
leukocytes increased to 13,000 and later to 20,000, to fall again fol- 
lowing the removal of the obstruction. In a second case operated 
by Cushing there was a persisting hyperleukocytosis, associated 
with abdominal pain and tenderness, at one time reaching 15,200. 
Upon the development of general peritonitis the count showed only 
4300. Cabot remarks, "Steadily increasing . leukocytosis is always 
a bad sign, and should never be disregarded, even when other bad 
symptoms are absent," to which Cushing adds, "A decreasing leuko- 
cytosis may be a much worse sign" (Finney 1 ). 

In paratyphoid fever the blood condition is essentially the same as 
in typhoid fever, viz., hypoleukocytosis early in the disease, disap- 
pearance of the eosinophiles, and later a marked increase of the lymph- 
ocytes which continues well into convalescence. 

Measles 2 is the second notable exception to the general rule that 
the acute infections are associated with a polynuclear neutrophilic 
hyperleukocytosis. But it is interesting to note that here also the 
hypoleukocytosis is preceded by a preemptive hyperleukocytosis, 
which commences at the beginning of the period of incubation, then 
increases rapidly and reaches its maximum about the sixth day be- 
fore the appearance of the eruption. After this it diminishes, and 
at the appearance of the exanthem and during its course the occur- 
rence of an increased number of leukocytes indicates some compli- 
cation. The hypoleukocytosis affects the polynuclear neutrophiles 
both absolutely and relatively, while the lymphocytes are relatively at 
least increased. The eosinophiles disappear. The hypoleukocytosis 
generally reaches its maximum on the second day of the eruptive 
stage, when the number of leukocytes is reduced to about one-half. 
After this they increase again more or less rapidly and reach the 
normal one to five days after the disappearance of the rash, unless 
some complication should supervene. Numerous eosinophiles then 
appear together with an absolute and relative increase of the poly- 
nuclear neutrophiles. 3 Manicatide and Galasescu 4 do not share 
the generally accepted idea of the decrease of the leukocytes during 
the eruptive stage of measles. They maintain that a mild increase 
is usual during this time, which then disappears with desquamation. 

Urticaria, syphilitic roseola, scarlatina, and the exanthem which may 
follow antitoxin treatment are not associated with hypoleukocytosis. 

1 Surgical Treatment of Perforating Typhoid Ulcer. Studies in Typhoid 
Fever. Johns Hopkins Press, 1901, p. 170. 

2 Reckzeh, Zeit. f. klin. Med., vol. xlv, p. 107 (full literature). 

3 Renaud, These de Lausanne, 1900. 

4 Folia haematol, vol. i, p. 110. 



100 THE BLOOD 

In uncomplicated cases of tuberculosis there is usually no increase 
of the leukocytes; when it does occur it is generally referable to 
suppurating cavities, recent hemorrhages, and resulting anemia, or 
to advancing pneumonia. The increase which occurs under such 
conditions is moderate and does not often exceed 15,000 cells. Ewing 
states that he has seen both lungs consolidated and riddled with 
small cavities in a case lasting but five weeks, and jet the leukocytes 
were never found above 12,000. He suggests that the absence 
of leukocytosis in such cases of acute phthisis which resemble pneu- 
monia may often be of value in diagnosis. Unfortunately there 
is no large series of examinations available from which to decide the 
relative value of the morphological examinations of the blood in the 
differential diagnosis between acute miliary tuberculosis and typhoid 
fever. According to Cabot and Warthin, a subnormal number of 
leukocytes may also be observed in acute miliary tuberculosis, while 
Kolner 1 thinks the leukocyte count important in distinguishing 
between the two diseases. 

In Malta fever the number of the leukocytes is usually not increased; 
some authors report that there is hypoleukocytosis as in typhoid fever. 2 

In uncomplicated cases of cachexial fever (Kala-azar) the leuko- 
cytes according to Rogers are markedly decreased. In India a 
number smaller than 2000 is regarded as almost diagnostic of the 
disease, but this may occur also in the true malarial cachexia. Rogers 
regards a reduction of the ratio of the whites to the reds to below 
1 to 1500 as quite characteristic of cachexial as compared with other 
Indian fevers. The more marked the hypoleukocytosis and the 
reduction of the neutrophiles the worse the outlook. The neutrophiles 
usually number from 40 to 50 per cent. 3 

Carpenter and Sutton report low values in dengue, viz., 4460 as 
average with 1886 as minimal. 4 

In uncomplicated cases of influenza the total number of leuko- 
cytes is commonly diminished; it may, however, be normal. When 
an increase beyond 15,000 occurs, some complication probably exists. 
During the course of the disease I have noted the existence of 
lymphocytosis, with low eosinophile values. During convalescence 
the neutrophiles may show maximal normal values. 

Hypoleukocytosis is one of the most constant symptoms of per- 
nicious anemia, during the active period of the disease. At times it 
may be extreme. Strauss and Rohnstein 5 cite two cases with 400 and 
328 cells respectively. As a rule it is much more moderate, viz., 
2000 to 3000 cells per cubic millimeter. 

1 Loc. cit., p. 96. 

2 E. Axisa, Centralbi. f. inn. Med., 1905, No. 11. 

3 Brit. Med. Jour., April 1, 1905. 

4 Jour. Amer. Med. Assoc, January 21, 1905. 
5 JL,oc. cit. 



MICROSCOPIC EXAMINATION OF THE BLOOD 101 

In splenic anemia also hypoleukocytosis is a common feature 
at some period in its course and is at times most marked. Osier 
mentions a case of Vickery's in which only 650 to 700 leukocytes were 
counted per cubic millimeter, and one of Peabody's with 800 cells. The 
average count in the series collected by Osier was 3850. * Immediately 
after a profuse hemorrhage or in a terminal infection there may be a 
hyperleukocytosis. 

In other types of severe anemia hypoleukocytosis is less constant. 

While the hypoleukocytosis in the diseases mentioned is rarely 
extreme, most extraordinary instances of leukopenia are at times 
encountered. Ehrlich 2 cites the case of a well-built young man 
in whom brief epileptiform seizures occurred, and in one of which 
the patient died. The postmortem examination was entirely nega- 
tive. During the three days preceding death two examinations 
of the blood were made. On the first not a single leukocyte could 
be demonstrated in ten blood films, and on the second day but one 
was found in the same number of specimens. 

Of drugs, atropin, camphoric acid, tannic acid, picrotoxin, agaricin, 
menthol, sulphonal, and several other antihydrotics, cause a marked 
decrease of the leukocytes. 3 

Neutrophilic Karyomorphism. — I have pointed out before that 
the neutrophilic elements of the blood can be subdivided into five 
classes according to the number of nuclear divisions (p. 81), and that 
the percentage figures representing these classes are normally quite 
constant for one and the same individual. Arneth has shown that in 
disease marked deviations from these normal standards may occur, and 
that the qualitative changes may be most pronounced even though there 
be no quantitative changes in the total number of the leukocytes, and 
vice versa. He accordingly distinguishes between, iso-, normo-, hyper-, 
and hypocytosis, and aniso-, normo-, hyper-, and hypocytosis, the term iso 
and aniso having reference to a normal or abnormal nuclear picture, 
respectively. Arneth's results are very interesting and show con- 
clusively that the absolute leukocyte count per se is relatively of 
little importance, and that a more detailed morphological study of 
the blood is necessary in order to derive all the information possible 
from the blood examination. I have myself insisted for years that of 
the two the differential count is more important and from my experi- 
ence with Arneth's nuclear studies I am quite prepared to admit 
that his method will at times furnish information of value, even when 
the differential count shows but little abnormality. 

When alterations in the nuclear picture do occur the change usually 
first affects the maturest forms, viz., group 5; then follow the others 
until in extreme cases the youngest forms largely remain. As aniso- 

1 Amer. Jour. Med. Sci., 1902, vol. cxxiv, p. 751. 

2 Die Ansemie, loc. cit. 

3 Bohland, Centralbl. f. inn. Med., 1899, No. 15. 



102 THE BLOOD 

hypocytosis, according to Arneth, represents the most serious condition 
so far as the leukocytic blood picture goes, as it indicates both an 
extensive destruction of leukocytes and a defective new formation. 
Less serious would be an anisonormocytosis, more favorable an 
anisohypercytosis, and most favorable an isohypercytosis. 

Anisohypocytosis occurs in fatal cases of pneumonia, con- 
stantly in typhoid fever and measles, frequently in varicella and 
mumps, further in severe cases of septicemia, in septic diphtheria, 
miliary tuberculosis, in acute articular rheumatism, fulminating 
appendicitis, and variola in the initial and eruptive stages. 

In malignant diseases, so long as no complications exist, no spe- 
cial influence upon the nuclear picture can be demonstrated. If com- 
plications or marked metastases occur corresponding changes occur. 

Polynuclear Eosinophilic Hyperleukocytosis (Eosinophilia). 1 — 
A physiological increase of the eosinophiles beyond the maximum 
observed in adults is seen in young children. According to Zap- 
pert, the relative numbers may here vary between 0.67 and 11 per 
cent., and Miiller and Rieder even speak of 21 per cent. Personally 
I am not prepared to admit that such high figures occur in normal 
children. I rather imagine that some of these instances were cases 
of worm infection. In older children normal adult values prevail, 
and it is then legitimate to consider an increase beyond these figures 
as abnormal. 

It is stated by some that there is a physiological increase of the 
eosinophiles during the menstrual period and following coitus. This 
is inconstant and rarely marked. 

Eosinophilia is thus essentially a 'pathological phenomenon. It 
occurs under the most diverse conditions, as in myeloid leukemia, 
in bronchial asthma, in various skin affections, the helminthiases, 
gonorrhea, osteomyelitis, following the injection of tuberculin, etc. 

In myeloid leukemia an absolute increase in the number of eosino- 
philes is one of the most constant symptoms. Ehrlich indeed has 
taught that this increase occurs in all cases and must be demonstrable 
to warrant the diagnosis. In view of recent advances in our knowl- 
edge of the pathology of the disease, however, this idea can no longer 
be upheld, as it has been shown that all forms of leukemia are or at 
least may be of myelogenous origin. 2 Cases have been recorded 
in which the blood picture was essentially that of the orthodox lym- 
phatic variety, but in which postmortem examination showed a 
total absence of involvement of the lymph glands, while the bone- 
marrow was extensively diseased. In these cases there was no increase 
in the total number of the eosinophiles. But it seems that even in 
those cases in which the blood picture is essentially that of a mye- 

1 For a thorough review of the literature on eosinophilia see C. E. Simon, 
International Clinics, 15th series, vol. iv. 

2 Pappenheim, Zeit. f. klin. Med., vol. xlvii, p. 216 



MICROSCOPIC EXAMINATION OF THE BLOOD 103 

lemia the usual increase in the number of the eosinophiles may be 
lacking. I have reported an instance in which these cells were not 
only not increased, but practically absent. 1 Such cases, however, 
are exceedingly rare, and it may still be regarded as the rule that in 
those cases of leukemia in which extensive myelocytosis exists the 
eosinophiles are absolutely if not relatively increased. With septic 
complications occurring in the course of the leukemias the eosino- 
philes are materially diminished, and in some cases they may be 
absent. Exceptions, however, occur, and Ehrlich cites a case in 
which the absolute number of eosinophiles Was still between 1400 
and 1500 per cubic millimeter, although the percentage had fallen 
from 3.5 to 0.43. 

In bronchial asthma an increase of the eosinophiles is observed 
quite constantly about the time of the paroxysm, and may amount 
to from 10 to 53.6 per cent. 2 Its occurrence is of value in differ- 
ential diagnosis, as renal, cardiac, and diabetic asthmas are not asso- 
ciated with eosinophilia. Between attacks the numbers may be 
normal or increased. 

In scarlatina? an increased number of eosinophiles is quite con- 
stantly observed at some time in the course of the disease. As the 
result of an anlaysis of 167 cases Bowie finds that at the onset of 
the fever they are diminished. In simple favorable cases they then 
increase rapidly until the height of the disease is passed, when they 
diminish again, and finally reach the normal some time after the 
general hyperleukocytosis has disappeared, viz., when the poison has 
all been eliminated. The more severe the case the longer are the 
eosinophiles subnormal before they rise again; in fatal cases they 
never rise, but rapidly decrease to zero. Bowie thinks that the 
curve of the eosinophiles is of value from a prognostic standpoint. 
If they are normal or subnormal after the first day or two, the case 
will in all probability be a severe one. In Reckzeh's series the 
highest percentage was 12.5, and the largest total number 1350. 

In measles an increase of the eosinophiles does not occur. 

In many skin diseases eosinophilia may also occur, especially 
in the bullous dermatoses, viz., Duhring's dermatitis herpetiformis, 
pemphigus foliaceus, and pemphigus vegetans, where 12 to 22 per 
cent, represent average values. In other skin diseases the tendency 
toward hypereosinophilia is also fairly pronounced, the degree of 
increase depending very largely, though not exclusively, upon the 
extent of the local process, as also upon the severity of the case. 
Light cases may thus show values which are practically normal. 
The list of diseases in which an increased eosinophile count has been 

1 C. E. Simon, Amer. Jour. Med. Sci., June, 1903. 

2 Billings, N. Y. Med. Jour., vol. lxv, p. 691. 

3 Zappert, Zeit. f. klin., Med., 1893, p. 292. Reckzeh, ibid., vol. xlv (literature). 
Bowie, Jour. Path. u. Bact., 1902, vol. viii, p. 82. 



104 THE BLOOD 

noted includes herpes zoster, prurigo, eczema (as high as 45 per cent.), 
psoriasis, lichen ruber planus, urticaria (up to 60 per cent.), derm- 
atoses of toxic origin — lead, mercury, picric acid, benzin (up to 31.5 
per cent.), sclerodermia, mykosis fungoides (37 per cent.), lupus and 
lepra (8 to 61 per cent.). Exceptions, however, also occur. 

Sabrazes and Mathis have described eosinophilia in connection 
with a disease termed Ki-Mo, occurring in Tonkin and Laos. 1 

Leredde and Poutrier have observed eosinophilia in association 
with a skin eruption following the ingestion of antipyrine. 2 

Of special interest is the increase of the eosinophiles in the helmin- 
thiases. This is particularly marked in ankylostomiasis (uncinariasis, 
hookworm infection), where 72 per cent, were counted in one case. 
As a general rule, however, it is not so extensive. The eosinophilia 
in hookworm infection is very constant and unless other manifest 
symptoms exist which would suggest a different origin, its occurrence 
should always lead to a careful examination of the stools for the eggs 
of the parasite. I have repeatedly made a probable diagnosis of 
uncinariasis in persons coming from the sand region of the South, 
on the basis of a blood hypereosinophilia, where no other clinical 
symptoms and no anemia existed, but where subsequent examination 
of the feces bore out the correctness of the diagnosis. The increase 
of the eosinophiles may be out of all proportion to the number of the 
eggs found. This method I should suggest especially when large 
bodies of men are to be examined. The fecal examination can then 
follow in those cases, where blood examination shows the existence of 
a definite anomaly. 

It is noteworthy that the hypereosinophilia of uncinariasis may be 
absent when the individual is greatly reduced by anemia. 

From a study of 100 cases of uncinariasis in Porto Rico, Ashford 3 
draws the following conclusions: 

1. Eosinophilia occurs at some period in all cases. It is most 
marked in early cases and in late cases where blood regeneration is 
still active; 20 to 50 per cent, is then not uncommon. 

2. In chronic cases or in those who have been profoundly anemic 
for a long time the eosinophile count is more apt to be low than high. 

3. When there is a fall of eosinophiles, accompanied by a lack of 
improvement in the physical signs, death is apt to follow. A slow 
rise in eosinophiles marks a long convalescence. 

In bilharziasis eosinophilia is also well pronounced. In 22 pure 
cases, uncomplicated by uncinariasis, reported by Kautsky-Bey, 4 the 
minimum percentage was 5 and the maximum 53. In the majority 
of cases the values were between 10 and 20. 

1 Gaz. hebd. de Bord., 1903, p. 182. 

2 Soc. de biol., 1903. 

3 Amer. Med., 1903. 

4 Zeit. f. klin. Med., 1904, vol. lii, p. 192 



MICROSCOPIC EXAMINATION OF THE BLOOD 105 

In the presence of oxyurides Buckler 1 found 16 per cent.; 19 per 
cent, were counted in association with ascarides, and Leichtenstern 
reports one case of Taenia mediocanellata with 34 per cent. It is to 
be noted, however, that eosinophilia is not a constant feature in infec- 
tions with the common taenia?, oxyuris, and ascaris, and that the number 
may not exceed minimum normal values. In cases of infection with 
the bothriocephalus eosinophilia does not occur (Schaumann). This 
has been the experience also of others, at least in those cases in which 
active and pronounced anemia existed. After removal of the worm 
the eosinophiles may, however, temporarily increase beyond the 
normal, as in one of Gilman Thompson's cases 2 (9 per cent.). 

In a fatal infection with Balantidium coli Strong and Musgrave 3 
observed a relative increase, and it appears that in amebic colitis also 
a moderate eosinophilia is not uncommon. 4 

As Brown 5 has shown, a remarkable increase of the eosinophiles 
occurs in trichinosis during the acute stage. In his first four cases 
with a total leukocyte count of 35,000, 13,000, 17,000, and 18,000 
the percentage of eosinophiles was 68.2, 42.8, 49, and 48, respect- 
ively. Kerr noted even a higher percentage in one case, viz., 86.6. 
Similar results have been obtained by Thayer, 6 Cabot, 7 Gwyn, 8 
Blumer-Neumann, 9 and others, and it can now be regarded as an 
established fact that the occurrence of eosinophilia is one of the 
most constant and diagnostically impoitant symptoms of the dis- 
ease. It is in a general way proportionate to the intensity of the infec- 
tion; when this is profound, however, the eosinophiles may not be 
increased and may indeed be diminished (Da Costa, Opie, Howard, 
Drake, and Cutler 10 ). The eosinophilia persists for a long time (in 
my own case for three months). A very interesting case of trichinosis 
is reported by McCrae, n in which the disease was complicated by 
typhoid fever; the eosinophilia was here nevertheless well marked. 

In filariasis also eosinophilia may occur. As the result of his 
study of four cases of the disease in the Philippines, Calvert 12 concludes 
that in the early stages hyperleukocytosis with an increase of the 
eosinophiles may be looked for, but that the number of the leukocytes 
in general, as also of the eosinophiles, returns to normal as the disease 
progresses. In one of his cases the percentage increased to 22, but 

1 Munch, med. Woch., 1894, Nos 2 and 3. 

2 Med. News, April 8, 1905. 

3 Johns Hopkins Hosp. Bull., ,1901. 

4 Amberg, "Amoebic Colitis in Children," Johns Hopkins Hosp. Bull., 1901. 

5 Jour. Exp. Med., vol. hi, p. 315; and Johns Hopkins Hosp. Bull., 1897. 

6 Phila. Med. Jour., vol. i, p. 654. 

7 Boston Med. and Surg. Jour., vol. cxxxvii, p. 676. 

8 Centralbl. f. Bakt., vol. xxv, p. 746. 

9 Amer. Jour. Med Sci., vol. cxix, p. 14. 

10 Trans. Assoc. Amer. Phys., 1902, p. 356. 

11 Amer. Jour. Med. Sci., 1902, vol. cxxiv, p. 56. 

12 Johns Hopkins Hosp. Bull., 1902, vol. xiii, p. 133. 



106 THE BLOOD 

varied within twenty-four hours between this point and 8. In a 
case of long standing which I had occasion to examine I found but 
2 per cent, of eosinophiles, with 36 per cent, of lymphocytes. Cal- 
vert, on the other hand, noted an increase of the lymphocytes. A 
relation between the number of embryos and the percentage of the 
different leukocytes does not appear to exist. 

In hydatid disease the number of observations so far recorded 
is as yet too small to indicate the frequency with which hypereo- 
sinophilia is observed. Seligman and Dudgeon 1 report a case of 
hydatid disease of the liver with 57 per cent, of eosinophiles. Bloch 2 
mentions one with 14.7 per cent., notwithstanding the fact that the 
cyst was suppurating. Still more lecently Sabrazes 3 described three 
cases, all with hypereosinophilia, and still others are recorded by 
Audibert, 4 Dargein and Tribondeau, 5 Achard and Clerc — one case 
with 40 per cent., 6 Achard and Laubry, 7 Frederici 8 and Meyer. 
That not all cases, however, are associated with hypereosinophilia is 
indicated by the negative reports of Bloch, Gourrand, 9 Launois and 
Weil, 10 and Besancon and Weil. 11 

Dr. J. Ramsey, of Launceston, Tasmania, has kindly sent me his 
findings in 5 cases of hydatid disease. In 4 of these there was no 
suppuration, but eosinophilia (28.4 per cent.) only occurred in 1. 
The others presented normal values; in 1 suppurating case no 
eosinophiles were seen. 

In Medina worm (Guinea worm) infection eosinophilia has also 
been noted. 12 It is said to be present always in connection with the 
fever due to the presence of the worm. 

It is generally stated that in malaria the eosinophiles are present 
in increased numbers during the afebrile period, and rarely diminish 
below the minimum normal values even at the time of a paroxysm. 
Zappert 13 reports a case in which on the day following the last attack 
20.34 per cent. (1486 absolute) were found. Krauss 14 on the other 
hand, in an analysis of 204 cases of malaria, in nearly all of which 
the organism could be demonstrated, found but 2 per cent, on an 
average and supernormal values only exceptionally. Fontaine, who 
has had extensive experience in the study of this question in Louisiana, 
tells me that in uncomplicated malaria eosinophilia is but rarely seen. 

In malignant disease eosinophilia apparently occurs in only a rela- 
tively small percentage of cases, and when present is usually of 

1 Lancet, June 21, 1902. 2 Deut. med. Woch„ ; No. 29, 1903. 

3 Gaz. hebdom. des sci. med. de Bordeaux, 1903. 

4 L'eosinophilie, Paris, 1903. 5 Soc. de biol., 1901. 
6 Gaz. hebdom., 1902. 7 Soc. de biol., 1901. 
8 Rivista crit. di clin. med., 1902. 9 Cited by Meyer (50). 

10 Soc. med. des hop., 1902. ll Arch. gen. de med., 1902. 

12 Remlinger, Soc. de biol., July 9, 1904. 

13 Zeit. f. klin. Med., vol., xxiii, p. 227. 

14 Jour. Amer. Med. Assoc, October 22, 1904. 



MICROSCOPIC EXAMINATION OF THE BLOOD 107 

moderate grade — i. e., not exceeding 7 to 10 per cent. Occasionally, 
however, the increase is most remarkable. Reinbach cites a case of 
lymphosarcoma (malignant lymphoma) of the neck with metastases 
in the bone-marrow, in which 60,000 eosinophiles were counted. 

In the differential diagnosis of carcinoma from pernicious anemia I 
have found that an increase of polynuclear neutrophiles associated 
with a normal or supernormal eosinophile count is very suggestive of 
cancer. 

A gonorrheal eosinophilia has been noted by various observers. 
From an analysis of 45 cases which Owings studied in my laboratory 
it appears that with an extension of the inflammatory process to the 
posterior urethra the number of cases increases in which an increased 
percentage of eosinophiles is found in the blood, and in cases of 
prostatitis eosinophilia is the rule. During the first week of the 
disease the blood is apparently always normal. In the second and 
third weeks it is normal in only 33 per cent, of all cases, and after 
two months' duration an increased number is still observed in 40 
per cent. The percentage of the eosinophiles usually does not 
exceed 12 per cent. At times, however, larger numbers are found; 
Bettmann cites a case of gonorrheal epididymitis with 25 per cent. 
Occasionally the eosinophilia is associated with a neutrophilic hyper- 
leukocytosis: this is usually of moderate intensity, but may be quite 
marked when the urethritis is complicated by an epididymitis, an 
orchitis, or a cystitis. 

More commonly the neutrophiles are diminished in uncomplicated 
cases while the lymphocytes and often also the large mononuclear 
leukocytes are increased. 

It is important to note that in gonorrheal arthritis also the eosino- 
philes are increased. 

In association with chronic tumors of the spleen and after extirpa- 
tion of the organ eosinophilia has been repeatedly observed. After 
extirpation eosinophilia is not immediately observed, however, but 
develops only after many months and is of moderate grade. 

Granasso 1 claims that in surgical tuberculosis of children the eosino- 
philes are always increased, the number being largest in the lighter 
and in the convalescent cases. In the event of a complicating febrile 
disease or in connection with pyogenic infections a drop, of course, 
takes place. In a suppurating glandular case I recently counted 12 
per cent. 

As I have pointed out, the eosinophilic leukocytes are relatively 
diminished and may disappear altogether in the great majority of 
the acute infectious diseases, with the exception of scarlatina, while 
hyperleukocytosis referable to the polynuclear neutrophilic cells exists. 
In the postfebrile period, however, the upper limit of the normal and 

1 Osped. Maria Vittorio, Torino. 



108 THE BLOOD 

even a well-marked eosinophilia are often observed. Turk 1 found an 
epicritic eosinophilia of 5.67 per cent. (430 absolute) in a case of pneu- 
monia, and after an attack of acute articular rheumatism 9.37 per 
cent. (970 absolute), I have seen an eosinophilia of 10.5 per cent, 
after pneumonia. 

An eosinophilia referable to drugs has been described, but has 
attracted little attention. Neusser mentions that pilocarpine will pro- 
duce eosinophilia, as also iron, nuclein, and its derivatives, but gives 
no details, v. Noorden reports the occurrence of eosinophilia follow- 
ing the internal use of camphor (in two chlorotics). Similar observa- 
tions have been made in animals after poisoning with carbon dioxide. 

Following the injection of tuberculin an increase of the eosino- 
philes has been observed in those cases in which a febrile reaction 
had occurred. In one case reported by Grawitz the eosinophilia 
reached its. highest point, viz., 41,000, three weeks after the injections 
had been stopped. 

Polynuclear Eosinophilic Hypoleukocytosis (Hypo-eosino- 
philia). — A diminution in the number of the eosinophiles is notably 
observed in the acute infectious diseases which are associated with a 
neutrophilic hyperleukocytosis. The only exception to this rule appar- 
ently is scarlatina, but here also their number is diminished at the 
onset of the fever, and, as I have stated, in fatal cases they rapidly 
disappear. Aside from the infections which lead to an increase 
of the polynuclear neutrophiles, hypo-eosinophilia also occurs in 
those forms which, like measles and typhoid fever, are associated 
with a decrease of the leukocytes. We may accordingly formulate 
the general rule that a diminution in the number of the eosinophiles 
will be observed at some period in the course of the various acute 
infectious diseases, no matter whether they are associated with a 
general polynuclear hyperleukocytosis or not. The extent to which 
this may go is variable; in the milder infections the values may be 
but little, if any, below the minimum normal, but in the severer and 
more protracted cases not a single eosinophile may be met with in a 
differential count of a thousand. Whether or not cases occur in 
which they are wholly absent I am not prepared to say. I have 
pointed out before that I designate a neutrophilic increase associated 
with an eosinophilic decrease as the septic factor, and regard its 
demonstration as one of the most valuable symptoms in the diagnosis 
of pyogenic infections. In active appendicitis it is of constant occur- 
rence and will serve to differentiate the condition from non-infectious 
abdominal affections. 

Aside from the acute infectious diseases it is uncommon to meet 
with a material diminution of the eosinophiles. It has been observed 
after severe muscular exercise and after castration, and it is com- 

1 Klinische Blutuntersuchungen, Wien, 1898. 



MICROSCOPIC EXAMINATION OF THE BLOOD 109 

monly noted in lymphatic leukemia with high lymphocyte counts. 
Da Costa 1 states that he has found a decrease or even an absence 
of eosinophiles in the majority of cases of chlorosis and pernicious 
anemia. This decrease in pernicious anemia has also been observed 
by others, 2 and is apparently the rule during the active stage of the 
disease ; in the interval, however, normal and even supernormal 
values may be obtained. It is essentially seen in the kryptogenetic 
type, while in parasitic pernicious anemia the eosinophiles may be 
increased. In these cases a decrease may, however, also occur when 
the infection is very severe. 

I have reported a remarkable case of atypical myeloid leukemia in 
which eosinophiles were practically absent. 3 ■ 

Lymphocytosis. — According to Ehrlich's conception of lympho- 
cytosis as a passive hyperleukocytosis, an increased number of lympho- 
cytes will be found in the blood in conditions which are associated 
with a hyperplasia of lymphadenoid tissue, the lymphocytes being 
mechanically washed into the blood current. But, as I have pointed 
out, there is evidence to show that the lymphocytes also may follow 
the laws of chemotaxis, and that an active lymphocytosis may possibly 
occur which is analogous to the hyperleukocytoses referable to the 
polynuclear granular elements. 4 

Under physiological conditions an increased number of lympho- 
cytes is notably observed in early childhood. Following the tem- 
porary increase of the polynuclear neutrophiles which occurs during 
the first twenty-four hours, the lymphocytes rapidly increase in 
number, so that by the twelfth day they represent 45 per cent, of 
all leukocytes (Carstanjen). Gundobin gives 59 per cent, as an 
average value for sucklings as compared with 34.6 per cent, of poly- 
nuclear neutrophiles. In adult life a physiological increase of the 
lymphocytes is notably seen in connection with the increase of the 
polynuclear neutrophiles which occurs during the process of digestion. 

Under pathological conditions lymphocytosis is more common in 
children than in adults, and it is noteworthy that in anemic and 
poorly developed children the normal ratio of lymphocytes to the 
polynuclear neutrophiles is reached only late. As a general rule the 
increase of the lymphocytes is not excessive and does not raise the 
total leukocyte count much above 30,000 to 40,000. Lymphocytosis 
of this order is notably seen in rickets, in whooping-cough, measles, 
congenital syphilis, in various subacute intestinal disorders of child- 
hood, at times in bronchopneumonia, etc. 

1 Clinical Hematology, Blakiston, Phila., 1901. 

2 Strauss u. Rohnstein, loc. cit., p. 31. 

3 Simon, Amer. Jour. Med. Sci., June, 1903. 

4 Jolly, "Sur les mouvements amoeboides des globules blancs," etc., Comptes- 
rend. de la Soc. d. biol., 1898, vol. x, serie v; and Wolff, "Giebt es eine aktive 
Lymphocytose," Deutsch. Aerzte-Zeit., 1901, No. 18, 



110 THE BLOOD 

In whooping-cough during the convulsive stage the total number 
of leukocytes may be increased to four times the normal; the average 
in De Amicis and Pacchioni's series 1 was 17,943. According to these 
observers, the hyperleukocytosis is demonstrable on the first day of 
the disease; it reaches its highest point in the convulsive stage and 
persists some time after cessation of the typical symptoms. Wanstall 2 
in his series of 16 cases finds no evidence of a marked general hyper- 
leukocytosis, and reports that in some the leukocytes were actually 
decreased. He could demonstrate a well-marked lymphocytosis dur- 
ing the catarrhal stage, however, in almost every case, which varied 
between 40 and 60 per cent. Wanstall concludes that an increased 
percentage of lymphocytes, at least equalling if not exceeding that of 
the polynuclear neutrophiles, is a valuable aid in the diagnosis of 
whooping-cough before the characteristic symptoms of the disease 
have appeared. Exceptions, however, occur, in which the lympho- 
cytosis does not reach the usual high figures. 

In rickets a well-marked lymphocytosis is the rule, which is both 
relative and absolute; the same holds good for congenital syphilis 
and for the secondary stage of the acquired disease. 

In bronchopneumonia there is at times a well-marked lympho- 
cytosis instead of a polynuclear hyperleukocytosis. Cabot cites an 
instance with a total leukocyte count of 94,600 and 66 per cent, of 
lymphocytes. 

In measles there is at first an increase of the polynuclear neutro- 
philic elements; later the lymphocytes increase in inverse proportion 
to the neutrophiles, the total number being largely dependent upon 
the degree of glandular involvement. 

In typhoid fever a relative lymphocytosis begins about the end of 
the first week and reaches its highest point in the stage of defer- 
vescence. Ewing states that he has found a uniform relation in 
this disease between the lymphocytosis in the blood and the grade of 
lymphatic hyperplasia found at autopsy. He records an instance in 
which the examination of the blood led to a strong suspicion of lym- 
phatic leukemia, and in which at autopsy the mesenteric glands were 
of unusually large size, and the edges of the partly necrotic intestinal 
ulcers rose 1.5 cm. above the mucosa. 

In smallpox there is a general tendency to an increase of the mono- 
nuclear elements. The same is seen in varicella. 

In tuberculosis, when well advanced, the lymphocytes are usually 
diminished, and the more so the more prominently the patients have 
become septic. Early in the disease and in convalescent cases there 
is often a distinct tendency to lymphocytosis. In this respect my 
observations agree very well with those of Holmes. 3 In a series of 202 

1 Clin. Med. Ital., 1899, No. 1. 

2 Amer. Med., 1902. 

3 Jour. Amer. Med, Assoc, Jan. 28, 1905. 



MICROSCOPIC EXAMINATION OF THE BLOOD HI 

cases he found, associated with a lymphocyte count of 10 or lower, only 

9 cases which could be viewed as recoveries or convalescents; with 

10 to 20 per cent., 14 cases, and with more than 20 per cent., 39 
cases. 

In uncomplicated influenza lymphocytosis is the rule during the 
active period of the disease, while in convalescence the neutrophiles 
may show maximum normal values. 

In epilepsy there may be distinct hyperleukocytosis, referable to an 
increase of the small mononuclear elements. Boston and Pearce 
found the neutrophiles down to 29 per cent. 

In pellagra mononucleosis apparently occurs with characteristic 
regularity, which may be of service in the diagnosis of the disease 
from other erythemas. 1 

In leprosy both lymphocytes and large mononuclear elements, but 
especially the former, are increased. 

A relative as well as absolute lymphocytosis occurs in the helmin- 
thiases in which the eosinophils are markedly increased. It is espe- 
cially pronounced in trichinosis. 

In general paresis, during the first stage, there is a tendency to 
hyperleukocytosis of the neutrophiles (70 to 80 per cent.); but in the 
third stage the latter fall as low as 40 per cent, while the lymphocytes 
are increased. 2 

A well-marked lymphocytosis is seen in Kala-azar. 

According to Sahli a decrease of the total leukocytes, associated 
with a relative increase of the lymphocytes, may be observed in hemo- 
philia. 

In uncomplicated cases of pseudoleukemia an absolute increase of 
the leukocytes does not occur; but there is usually a relative increase 
of the lymphocytes of such extent that the normal ratio to the poly- 
nuclears 1 to 3 rises to 2 to 3 to 1. This relative lymphocytosis Ehr- 
lich and Pinkus regard as characteristic of true pseudoleukemia, 
in the differential diagnosis from sarcomatosis and other lympho- 
matous growths. 3 

Grawitz, 4 on the other hand, maintains that from the leukocyte 
count no diagnostic conclusions can be drawn, and cites cases in 
which the ratio was either normal or in which the lymphocytes were 
actually diminished. 

When the pseudoleukemic process involves the bone-marrow the 
blood findings may be very variable. As a result of stimulation of the 
myeloid tissue myelocytosis may occur; in other cases the blood pic- 
ture closely resembles that of lymphatic leukemia (leukanemia, 

1 Grigorescu and Galasescu. Spitalul, 1903. 

2 Bruce, Scott. Med. and Surg. Jour., June, 1903. 

3 Pinkus, Die Leuksemie, Nothnagel's Encykl. 

4 Klinische Pathol d. Blutes, 2d ed. 



112 THE BLOOD 

pseudopernicious anemia). In all such cases anemia is at the same 
time a prominent symptom owing to the replacement of the ery- 
throblastic by lymphadenoid tissue. 

The highest grade of lymphocytosis is met with in the so-called 
lymphatic form of leukemia. As in the myeloid variety, the total 
number of leukocytes is here also very much increased, though 
not to the same extent. The highest count in Cabot's series was 
220,000 and the lowest 40,000, so that we may regard 130,000 as 
an average. The lymphocytes usually number more than 90 per cent. 
In the chronic cases the small lymphocyte prevails, while in the 
acute cases the large lymphocyte controls the blood picture. When 
septic complications develop, the total number of the leukocytes, 
as in the myeloid form of leukemia, likewise undergoes a considerable 
diminution, but the lymphocytes still remain relatively increased. 
In one of Cabot's cases, in which as the result of septicemia the total 
number of leukocytes fell to 471 per cubic millimeter, the percentage of 
lymphocytes still was 94.7. 

In the majority of cases of chloroma there is a moderate leukocyto- 
sis with lymphocytosis of the small or large cell variety, but in others 
myelocytes enter more or less prominently into the blood picture. 

An experimental lymphocytosis has been observed following the 
injection of tuberculin and of extract of carcinomatous tissue (Gra- 
witz). Waldstein claims to have produced a marked increase of the 
lymphocytes by hypodermic injections of pilocarpine, but, according 
to Ewing, this increase is only relative and brought about by a 
diminution of the polynuclear cells. Wilkinson speaks of a lymph- 
ocytosis following injections of quinine hydrochlorate and Perry 
has noted the same after the administration of thyroid extract. 1 

Lymphopenia. — Lymphopenia is notably observed in the acute 
infections which are associated with an increase of the polynuclear 
neutrophiles, and is almost always relative. The condition per se 
has received but little attention, and is relatively unimportant from the 
clinical standpoint. 

Variations in the Number of Large Mononuclear Leukocytes.— 
Variations in the number of the large mononuclear leukocytes are 
as a rule not sufficiently marked to cause either a distinct increase 
or decrease of the total number of the leukocytes. One notable 
exception to this rule, however, exists in the cases of the acute type 
of lymphatic leukemia, in which the predominant cell is the large 
lymphocyte, viz., the juvenile form of the common large mononuclear 
leukocyte, in the sense of Pappenheim. At the same time it must 
be noted that some cases of chronic lymphatic leukemia also occur 
in which the large mononuclear leukocyte and Ehrlich's transition 
form represent a large percentage of the leukocytes. These relations, 

1 Cited by Da Costa. 



MICROSCOPIC EXAMINATION OF THE BLOOD 1 13 

however, are not constant. A decrease is notably seen in myelocytic 
leukemia. 

In the so-called pseudoleukemia infantum of v. Jaksch a marked 
increase of the mononuclear elements is observed in a certain per- 
centage of cases, but in the larger number the general increase of 
the leukocytes is referable to an increase of the polynuclear cells. 

A relative as well as an absolute increase of moderate grade is 
observed in many of the diseases in which the lymphocytes are 
increased, as in rickets, syphilis, measles, scarlatina, smallpox, and 
according to my experience also in typhoid fever, etc. I have ob- 
served a marked increase in a case of Addison's disease a few days 
before death, and found notable numbers in debilitated individuals, 
in association with sloughing epithelioma, etc. 

In a fatal case of epidemic cerebrospinal meningitis with a high 
leukocytosis and polynucleosis which I recently saw there was both 
a relative and absolute increase of the mononuclear leukocytes. 
Some of these as well as some of the neutrophiies contained menin- 
gococci. 

In mycosis fungoides an increase of the large mononuclear elements 
has been noted by Hodara 1 and Pappenheim. 2 

A distinct increase is further observed in chronic malaria. In 
this connection Krauss 3 remarks that it is not so much the absolute 
increase of these cells which is diagnostic of malarial infection as the 
relative increase over the small mononuclears. In cases of malaiial 
infection without much fever and without quinine the polynuclears 
are at the same time markedly diminished, while during the rise of a 
malarial fever, or as a result of quinine therapy, the polynuclear 
neutrophiies may reach 80 per cent. ; but even then the large mononu- 
clears exceed the small mononuclears. 

In Kala-azar, as in malaria, there is a distinct tendency to an in- 
crease of the large mononuclears. In a series of 10 cases reported by 
Donovan, the average was 21.58 with variations from 6 to 48 per cent. 

Variations in the Number of the Mast-cells. — A small number 
of mast-cells is found in the blood under normal conditions. The 
presence of more than 1.5 per cent, is probably always pathological. 
A remarkable increase is noted in the myeloid type of leukemia 
and is one of the most constant features of the disease; more constant, 
in fact, than the increase of the eosinophiles. The percentage is not 
necessarily above normal, but not infrequently values of from 5 to 10 
per cent, are found. It is noteworthy that this increase of the mast- 
cells may be demonstrable at a time when the disease is apparently 
quiescent. In one instance of this kind the total number of the leuko- 

1 Monatsheft f. prakt. Dermat., 1904. 

2 Folia hsemat., vol. i, p. 487 

. 3 Jour. Amer. Med. Assoc, October 22. 1904. 



114 THE BLOOD 

cytes had been 350,000; three months later I counted but 2080, of 
which 10.9 per cent, were mast-cells, and later they rose to 15 per cent. 

The only other condition in which I have found such high values 
occurred in a patient, following fracture of the ankle and consequent 
cellulitis. In this case they rose to 17 per cent, and the blood in general 
presented a typical leukemic picture. A few days later normal 
values were found. 

A more moderate increase is noted in many other diseases. Gen- 
erally speaking, my experience has been that they are more numerous 
in conditions in which the eosinophiles also are increased, and are 
generally diminished when the eosinophiles are below normal. This 
rule, however, is not absolute. I have found values above the normal 
in various skin diseases, in gonorrhea, in certain cases of malignant 
disease, associated with eosinophilia. In one case of renal carcinoma 
a few weeks after the removal of the growth I counted more than 
2 per cent, of mast-cells, with but 1.9 per cent, of eosinophiles. 

Canon reports an increase of mast-cells in chlorosis; Sherrington, 
in cases of Asiatic cholera, dying in the reactive stage; Taylor, 
in two cases of septic bone disease ; Da Costa states that an increase 
has also been observed in some cases of splenic anemia. 

I have found the number diminished or entire absence of mast- 
cells in some cases of malignant endocarditis, appendicitis, empyema, 
influenza, tonsillitis, intestinal obstruction, lumbar abscess, periproc- 
titic abscess, pernicious anemia, hematoma of the abdominal walls, 
diabetes, carcinoma of the cervix (septic), "black" jaundice, pneu- 
monia (unresolved). In malaria the number is usually normal. 

Myelocytosis. — At birth and during the first weeks of life it is 
usual to meet with a small percentage of neutrophilic myelocytes in the 
circulating blood under perfectly normal conditions, while in adults 
their presence is always abnormal. In children the tendency to mye- 
locytosis is always more pronounced than in adults. Zelanski 
and Cybulski, 1 who have studied this question more particularly, have 
found myelocytes in a great many diseases. In the pseudoleukemia 
of v. Jaksch the number varied between 1.5 and 17.4 per cent., in 
congenital debility from 3.5 to 12.5 percent. In congenital syphilis 
they found myelocytes quite commonly, the number — usually moderate 
— depending upon the intensity and duration of the morbid process. 
They disappear upon institution of mercurial treatment. In rickets 
the number of myelocytes is dependent upon the intensity of the 
morbid process. In the lighter cases they are scanty or absent. 
Scrophulosis is not associated with myelocytosis. Tuberculosis and 
catarrhal processes involving the digestive apparatus, of long duration, 
cause the appearance of myelocytes in fairly large numbers. Very 
curiously acute dyspeptic processes were also found associated with 

1 Jahrb. f. Kinderheilk., 1904, vol. lx, p. 884 



MICROSCOPIC EXAMINATION OF THE BLOOD H5 

myelocytosis in very young children. In a case of congenital atresia 
of the anus they found 20 per cent, of myelocytes. 

Turk has shown that they are quite common in the acute infectious 
diseases of childhood, and in diphtheria Engel ascertained that 
they are especially numerous in the severe cases (3.6 to 16.4 per 
cent.). In mild infections they are not usually seen, and when 
present they are found in only very small numbers. In pneumonia 
they are absent or very few in number at the beginning of the dis- 
ease, while at the time of the crisis or immediately thereafter they 
become more numerous and in some cases represent 12 per cent, of 
all neutrophilic cells; such high percentages, however, are rather 
uncommon and are more apt to be encountered in children than in 
adults. In acute septic conditions a small number of myelocytes 
may also be observed; larger numbers are found in the more chronic 
cases, which are associated with marked anemia. In a case of 
lumbar abscess which had been discharging for six months I found 
7.8 per cent. 

In a case of "black" jaundice I found 2.2 per cent. Neusser has 
noted their presence in asphyxia and acute mania; Ewing states that 
they have been found in considerable numbers in rickets, osteomyeli- 
tis, and osteomalacia. Da Costa speaks of their occurrence in poison- 
ing by carbon monoxide, in hepatic cirrhosis, acute, gout, malignant 
endocarditis, and exophthalmic goitre. 

The occurrence of myelocytes under these conditions is to be 
regarded merely as a quantitatively or gradually increased polynu- 
cleosis of the corresponding granular cells, the result of an increased 
destruction of the adult cells and consequent increased production 
(anisohypercytosis). This is in contradistinction to the myelocytosis 
associated with myeloid leukemia where there is a primary increased 
formation referable to myeloid hyperplasia; this form is essentially a 
passive myelocytosis, while the former is active. 

In anemic conditions of whatever origin it is common to meet 
with a moderate number of neutrophilic myelocytes. In pernicious 
anemia they are quite constant in the active stage of the disease; 
as a rule the values do not exceed 0.5 to 1 per cent., but at times they 
may reach 7 per cent. 

Pappenheim makes a distinction between primary hemophthisic 
pernicious anemia of the Biermer type and the form referable to 
bothriocephalus infection, on the one hand, in which myelocytes in 
his experience do not occur, and primary myelophthisic spleno- 
medullary anemia, myelomatosis, and myelogenous pseudoleukemia 
(tumor anemia, pseudopernicious anemia) on the other, in which 
they may be present. 

According to Kurpjuweit 1 the occurrence of myelocytes in large 

1 Deutsch. Arch., vol. lxxvii. 



116 THE BLOOD 

numbers (4 to 17 per cent.) in connection with the symptom complex 
of a severe anemia is to be viewed as almost pathognomonic of malig- 
nant growth with bone-marrow metastases, even when a primary 
tumor cannot be found. 

In the secondary anemia associated with syphilis and malignant 
disease, as also in the pseudoleukemia of v. Jaksch, variable figures are 
found (1.5 to 17 per cent.). In a young child in which a notable 
anemia had developed as the result of amebic dysentery, Amberg 
counted 9 per cent. In the estivo-autumnal type of malaria they 
are quite common. 

The neutrophilic myelocytes which are met with under these 
various conditions are almost without exception of the small trachy- 
chromatic variety. The amblychromatic variety is practically only 
encountered in the myeloid type of leukemia, which is really the 
one disease in which large numbers of myelocytes of all kinds find 
their way into the blood. Upon their presence in numbers exceeding 
those found in all other diseases the diagnosis is largely dependent. 
The blood state is that of a true myelemia. The number of neutro- 
philic myelocytes in myeloid leukemia is often most remarkable, and 
a count of from 50,000 to 100,000 per cubic millimeter is by no means 
exceptional. The average percentage of 18 cases reported by Cabot 
was 37.7, corresponding to a total number of 162,000 leukocytes. 
Coincidently with the neutrophilic myelocytes eosinophilic myelocytes 
also appear in the blood and may constitute the majority of the eosino- 
philic cells seen in this type of the disease; their percentage, how- 
ever, is rarely large. The total number of the polynuclear eosino- 
phils is at the same time increased, although the relative percentage 
may be normal or even slightly below normal. The polynuclear 
neutrophilic cells and- the lymphocytes, while absolutely increased, 
are relatively much diminished. Of the latter, only 7.6 per cent, 
are found on an average, and of the former 49.2 per cent., as com- 
pared with 20 to 30 and 60 to 70 per cent., respectively, under nor- 
mal conditions. The mast-cells, as I have pointed out, are inva- 
riably present in increased numbers in the myeloid type of the disease. 

While the majority of the neutrophilic and eosinophilic cells 
present a normal habitus, it is common in myeloid leukemia to 
meet with dwarfed forms. Occasionally also leukocytes are ob- 
served which are undergoing mitosis. Of special interest is the 
fact that in certain chronic cases of the disease the neutrophilic 
cells apparently lose the power of forming neutrophilic material. 
Non-granular polynuclear cells and myelocytes then appear in the 
blood and may give rise to much confusion. In one case of this kind 
reported by Ehrlich the great majority of the mononuclear elements, 
which constituted 70 per cent, of the total number, were entirely 
free from neutrophilic granules. 

The total number of the leukocytes in myeloid leukemia in the 



MICROSCOPIC EXAMINATION OF THE BLOOD H7 

active stage of the disease is much increased. In Cabot's series 
of 30 cases the average was 438,000. If at the same time, as not 
infrequently occurs, there is a coincident anemia with marked diminu- 
tion of the red cells the ratio between the whites and reds may fall 
to 1 to 2 or even 1 to 1 ; there are cases on record, indeed, in which 
the leukocytes outnumbered the red cells. Formerly much stress 
was laid upon this ratio in the diagnosis of the disease; leukemia 
was regarded as a hyperleukocytosis in which the ratio exceeded 
a definite proportion that was generally placed at 1 to 50. As a matter 
of fact, there is probably no other disease in which so great an increase 
of the leukocytes is observed, and even at the present day the diagnosis 
is usually justifiable when an increase of such proportions is noted. 
But, as I have pointed out, myeloid leukemia is essentially a myelemia 
and not a hyperleukocytosis. There are cases, moreover, exceptional 
to be sure, in which the increase of the leukocytes is not so extreme. 
I have observed one case in which the total number was only 2080 
and the ratio of the whites to the reds 1 to 1015. The diagnosis of the 
disease should hence be based primarily upon qualitative changes in 
the morphology of the blood and only secondarily upon an increase 
of the leukocytes as a whole. 

When septic complications supervene in the course of the disease 
the blood condition may undergo marked changes. Thus, in propor- 
tion to the degree of infection the myelemic picture gradually dis- 
appears and is replaced by that seen in simple septic conditions. 
The polynuclear neutrophiles may then increase to 90 per cent., 
and even more, while the eosinophiles diminish and may almost 
disappear. 

X-ray treatment in a certain number of cases may cause a marked 
change in the blood picture. The total number may fall rapidly 
and there is a general tendency to normal conditions; a complete 
disappearance of myelocytes is, however, very rare. The mast-cells 
very curiously remain above normal. 

In the purely lymphatic form of leukemia neutrophilic myelocytes 
are scanty; there are cases of mixed leukemia, however, in which 
at some stage of the disease the blood picture is essentially of the 
lymphatic type, while at another period there is a marked myelo- 
cytosis. 1 

In a case of compound fracture with consequent cellulitis I found 
a blood picture which was typical of myelocytic leukemia, with large 
numbers of myelocytes (15 per cent.). After a few days there was a 
return to normal. Hastings tells me that he has found myelocytes 
in 3 to 7 per cent, of cases of fracture. 

1 For a detailed consideration of the blood changes in leukemia see especially : 
Pinkus, " Die Leukaemie," Nothnagel's Encycl. Ewing, Clinical Pathology of 
the Blood, Lea Brothers. Cabot, Clinical Exam, of the Blood, Wm. Wood & Co. 
Pappenheim. Zeit. f. klin. Med. Haematologische Streitfragen, 1903. 



118 THE BLOOD 

In pseudoleukemia myelocytes are essentially seen in cases where the 
pathological process has affected the bone-marrow (myeloid pseudoleu- 
kemia) and where, as a result of lymphadenoid substitution of the 
myeloid tissue, an irritative myelocytosis develops. 

Eosinophilic myelocytes, aside from their occurrence in myeloid 
leukemia, are comparatively rare. They have been found in the 
pseudoleukemia of infants; Mendel 1 speaks of their occurrence in 
a case of myxedema; Turk 2 reports that they are occasionally seen 
in some of the infectious diseases, and Bignami claims to have seen 
them in pernicious malaria. In one case of posthemorrhagic 
anemia referable to a ruptured tubal pregnancy I found 1 per cent, 
of eosinophilic myelocytes, and in a case of myelogenous leukemia, 
in which the eosinophiles were absolutely much diminished, the only 
eosinophile that I could find in many slides was a myelocyte. In a 
case of trichinosis they were also occasionally seen at a time when 
the eosinophiles were much increased. 



The Plaques. 

In addition to the leukocytes and red corpuscles large numbers of 
small, roundish elements are encountered in the blood which measure 
about 3 fJ. in diameter and are free from coloring matter (Plate II, 
Fig. 1). They are frequently seen collected into groups resembling 
bunches of grapes. These are the blood plates or plaques of Bizzozero. 
According to Hayem, they represent red corpuscles in an early stage 
of development, and'are themselves derived from leukocytes within the 
lymph channels. He terms them hematoblasts. This view is not 
shared by modern hemotologists. Lilienfeld, Hauser, Howell, and 
others regard the plaques as disintegration products of leukocytes, 
while still others look upon them as precipitated globulins derived 
in part from the morphological elements of the blood and in part 
originating directly in the plasma. More generally accepted is the 
view expressed by Engel, Bremer, Maximow, Pappenheim, and others, 
according to which the plaques are derived from the red cells by 
extrusion. They are originally contained in the interior of the cells 
as so-called nucleoids, and represent the remains of the original 
nucleus, which has lost its individuality as the result of chromatolysis. 
As a matter of fact, it is possible by suitable staining to demonstrate 
the plaques not only within the red cells, but also their extrusion 
from the cells, so that the erythroglobular origin of some of these 
formations at least can scarcely be doubted. Jost, moreover, has 
shown that in the blood of sheep and calf embryos they appear at a 

1 Berl. klin. Woch., 1896. No. 45. 

2 Klin. Untersuch. d. Blutes, etc., Wien u. Leipzig, 1898. 



MICROSCOPIC EXAMINATION OF THE BLOOD 1 ] 9 

time when leukocytes are not as yet demonstrable. But, on the other 
hand, there is a possibility that what we generally designate as plaques 
does not represent a unity, and that some of the elements which resem- 
ble the true blood platelets may be of different origin. To a certain 
extent such ill-defined little bodies are without doubt deriyed from 
leukocytes by a process of plasmorhexis — i. e., by the liberation of 
small bits of protoplasm. This may be observed under the microscope 
directly. 

Deetjen -has shown that the true plaques are capable of executing 
ameboid movements when the blood is placed on a slide which has 
been covered with a thin film of agar containing a certain amount 
of sodium chloride, sodium metaphosphate, and dipotassium phos- 
phate. He also believes to have demonstrated a nucleus in the in- 
dividual plaque, and concludes that the bodies in question do not 
represent artefacts or products of degeneration, but are true cellular 
elements. 

According to Osier, the number of plaques varies normally between 
200,000 and 500,000 per cubic millimeter. Brodie and Russell claim 
that this number is too small, and that with their improved method 
of counting an average of 635,300 is obtained. The normal ratio 
between the plaques and the red corpuscles would thus be 1 to 7.8, 
taking 5,000,000 as the average normal for the red cells. More recently 
Helber found variations between 192,000 and 264,000. 

Under pathological conditions the plaques may be increased or 
diminished. In pernicious anemia their number is very low; Van 
Embden found 64,000 and 32,000 in two cases. At times they are 
apparently absent, but in some cases increased numbers have been 
observed. 

According to Pappenheim the plaques are diminished in pernicious 
anemia owing to over-rapid maturation of the red cells. As a result 
the nuclei of the erythroblasts either do not become pyknotic and un- 
dergo chemical chromatolysis with consequent formation of oxyphilic, 
viz., azurophilic nucleoids, but are destroyed already in an early stage 
by karyorrhexis ; or, if they do become pyknotic, they are expelled 
from the cells plasmolytically in the anisotonic (anemic blood serum). 
A nucleoid thus does not remain which could later escape as a plaque. 

In leukemia the plaques are often greatly increased. A large 
increase is at times observed in posthemorrhagic anemia and in 
chlorosis, but the results are not constant. In the secondary anemias 
referable to carcinoma, sepsis, tuberculosis, etc., the findings are 
variable; sometimes an increase is observed, at others a decrease, and 
then again normal values; the results, moreover, are inconstant in one 
and the same case. In the acute infectious diseases their number 
is the smaller the more severe the course of the disease. In pneu- 
monia they are often diminished during the fever, but increased after 
the crisis. Similar results have been obtained in typhoid fever, while in 



120 THE BLOOD 

erysipelas they are found increased from the start. Enormous numbers 
of plaques may be seen in the course of trichinous infection. Schleip 
looks upon their appearance in large numbers as evidence of approach- 
ing convalescence. In my own case, however, they seemed to be 
most numerous at a time when the clinical symptoms were most 
active. 

(For the enumeration of the plaques see p. 144.) 

Literature. — Bizzozero, Virchow's Archiv, vol. xc. Hayem, Le sang, Paris, 
1889. Howell, Jour, of Morph., 1891, vol. iv, p. 57. Maximow, Arch. f. Anat., 
1899, vol. i, p. 33. Jost, Arch. f. mik. Anat., 1903, vol. lxi, p. 667. Determann, 
Deutsch. Arch. f. klin. Med., vol. lxi, p. 365. Deetjen, Virchow's Archiv, 1901, 
vol. clxiv, p. 239. Brodie and Russell, Jour. Physiol., 1897, Nos. 4 and 5. Heller, 
Deutsch. Arch., vol. lxxxi, Heft 3 u. 4. 



The Dust Particles or Hemokonia of Muller. 

These may be seen in any fresh specimen of blood mounted in the 
usual manner. They are small, generally round, sometimes dumb- 
bell-shaped, colorless, highly refractive granules, which manifest 
very active molecular movements. They occur in the plasma of the 
blood and are apparently not connected with the process of coagula- 
tion. Muller found them abnormally numerous in a case of Addi- 
son's disease, while they were diminished during starvation and in 
various cachectic conditions. Stokes and Wegefarth regard these 
granules as identical with the neutrophilic and eosinophilic granules 
of the leukocytes. They suppose that the bactericidal power of the 
leukocytes and of the serum of man and many animals is due to their 
presence. As a matter of fact, the origin of the hemokonia from 
the granular leukocytes can frequently be directly observed. 

I have quite constantly found the hemokonia increased at the 
height of digestion, and have then repeatedly observed their extru- 
sion from both neutrophilic and eosinophilic cells. 

Literature. — H. F. Muller, " Ueber einen bisher nicht beachteten Formbe- 
standtheil d. Blutes," Centralbl. f. allg. Path. u. path. Anat., 1896, p. 929 W. 
R. Stokes and A. Wegefarth, "The Presence in the Blood of Free Granules, etc., 
and their Possible Relation to Immunity," Johns Hopkins Hosp. Bull., 1897, 
p. 246. E. B. Sangree, " Leukocytic Granules," etc., Phila. Med. Jour., 1898, 
p. 472. 

General Technique. 

Slides and Cover-glasses. — To obtain satisfactory results, it is 
essential to have glassware of the best quality. The cover-glasses 
should not measure more than 0.08 to 0.10 mm. in thickness and must 
be cleansed with care. The same holds good for the slides, which 
should have a level surface; many of those furnished by dealers 
are unsatisfactory for work with immersion lenses. 



MICROSCOPIC EXAMINATION OF THE BLOOD 121 

Both covers and slides should be placed in concentrated sulphuric 
acid or in glacial acetic acid for several hours. They are thoroughly 
washed in running water and distilled water and then placed in 
alcohol and finally in ether, where they remain for several hours. 
During this process care must be had that they are well separated 
from each other. Subsequently they are kept in jars with absolute 
alcohol, and are dried just before use, or they may be dried at once 
with fine linen or Japanese lens paper and stored in dust-proof 
receptacles. When once cleansed, the cover-glasses should be 
handled only with forceps. 

To cleanse slides that have been used, the covers must first be 
removed by immersion for several days in xylol or turpentine. 
They are then placed in hydrochloric acid to which about a tea- 
spoonful of potassium chlorate has been added for every 30 c.c. 
The mixture is kept on the boiling water bath to the point of 
decolorization. The slides are next rinsed in hot water, heated for 
a half-hour in a thin mush of equal parts of washing soda, sawdust, 
and talcum, prepared with the aid of water and stirring frequently, 
then washed off with hot water acidified with hydrochloric acid, and 
finally with pure hot water, alcohol, and ether. 

The Blood Mount. — We distinguish between wet mounts and 
dry mounts. Wet specimens can only be utilized successfully if the 
patient is near at hand to the laboratory, as in office work and in the 
hospital; where several hours must elapse before the preparation can 
be examined, it will usually be best to resort to the dry specimen. 
Wet preparations, however, are very convenient and yield a large 
amount of information without delay, and a rapid survey will indi- 
cate whether or not it will be necessary or advisable to resort to a 
more detailed examination. The grade of an anemia: the degree, 
character, and extent of a hyperleukocy tosis ; the presence of malarial 
organisms, can all be told from the wet preparation. With the dry 
and stained specimen, on the other hand, all these points are brought 
out more distinctly, and other information is further afforded which 
cannot be obtained from the wet specimen alone. 

To prepare a blood specimen, the tip of a finger, or in children 
especially the lobe of the ear, is first cleansed with ether and then 
punctured with a suitable instrument, such as a fine lancet or a 
stout needle. The puncture should be sufficiently deep that the 
blood will flow from the wound without undue pressure. 

To prepare a wet specimen, a clean cover-glass is taken up with a 
pair of forceps, with flat blades and a light spring, touched to the 
drop without coming in contact with the skin, and immediately 
transferred to a clean slide. If suitable glassware is used that is 
perfectly clean, the drop will immediately spread out between cover- 
glass and slide, and on examining with a low power, which should 
always precede examination with a high power, it will be noted that 



122 



THE BLOOD 



in the central portion of the specimen especially the red cells will be 
well separated from one another and will not have run into rouleaux. 
This will only occur if the glassware is imperfect, if it is not perfectly 
clean, or if the drop has been too large. To gauge the proper size 
of the drop requires a little practice. Along the margin of the speci- 
men, where a certain amount of evaporation is going on, it is usual 
to find rouleaux and crenated red corpuscles, even though the rest 
of the specimen is perfect, and in the course of time postmortem 
changes will also become noticeable throughout the preparation. 
If the specimen is ringed with a little paraffin, however, a satisfactory 
examination is still possible after a number of hours, and even without 
being ringed such preparations can be kept for at least one hour. 

To prepare dry specimens, which are subsequently to be stained, 
the blood is spread between cover-glasses or on slides. 

Personally I have almost abandoned the use of cover-glasses, 
and much prefer slides for routine work. But little practice is 




Fig. 17. — The preparation of blood smears on slides. 

required to obtain very satisfactory results, and it is possible to control 
the quality of the individual smears with a degree of precision which 
is but rarely attained even by the most experienced workers with 
cover-glasses. The spreads, moreover, are much larger; so that 
there will always be a sufficient number of leukocytes available even 
under normal conditions to permit a count of at least a thousand 
cells. At the same time it is possible to spread portions of the drop 
so thin that the individual cells are well separated the one from the 
other, while other portions can be made a little thicker. The slides 
are cleansed in the same thorough manner as in the case of the cover- 
glasses. A fair-sized drop of blood is then mounted near the end of 



MICROSCOPIC EXAMINATION OF THE BLOOD 



123 



one slide and spread with an even sweep with the edge of a second 
slide; this should be done with a light hand, and holding the first 
slide in the left hand between the thumb and the second and third 
fingers. The second slide should also be held in this manner, but at 
an angle of 45 degrees to the first, as shown in the accompanying 
illustration (Fig. 17). Before commencing the sweeping movement 
I let the blood spread along the edge of the second slide by capillary 
attraction; then I move across, gradually raising the second slide to 
a vertical position. Pressure must be carefully avoided. 

If covers are to be used, one cover-glass is locked in a pair of 
forceps such as those devised by Ehrlich and pictured in the accom- 
panying illustration (Fig. 18). A second cover is taken up with 
a pair of forceps without a lock, but with flat blades and a light 
spring; this is held to the drop of blood just as it emerges from the 
puncture, and is then immediately laid upon the first cover. If the. 
glasses are of satisfactory quality and clean, the blood will at once 
spread in a capillary layer; the top cover is then drawn from the 
lower cover by grasping the edge firmly with the fingers and making 
even traction in a plane parallel to the other. Here also a certain 
amount of experience is necessary in gauging the size of the drop 




Fig. 18. — Ehrlich's cover-glass forcep 



in reference to the size of the covers. In no case should it be so 
large that the top cover floats upon the blood. If the drop is rather 
small, the two covers should overlap only to such an extent as to 
furnish a space which is just filled by the blood. If the drop is 
larger, they should overlap over a larger surface. 

After being allowed to dry in the air the specimens are placed 
between layers of filter paper and may then be stained at leisure. 
If several days must elapse before the examination, it is well to 
place them, wrapped in filter paper, in closed jars. Should it be 
desired to preserve the specimens for a long time — i. e., for months 
or years — it is best to coat the films with a thin layer of paraffin, 
which later is dissolved by immersion in toluol. In this manner 
especially valuable and rare specimens may be kept almost indefi- 
nitely. Unless this precaution is taken, the staining qualities of all 
the morphological elements of the blood will undergo changes which 
render the specimens unfit for color analysis. 

Fixation. — The selection of the method of fixation depends very 
largely upon the stain which is to be employed. If strongly alcoholic 



124 THE BLOOD 

solutions are used no previous fixation is necessary, but with aqueous 
solutions fixation must precede the staining. To this end several 
methods may be employed. The best results are obtained by heat. 
For this purpose a copper plate may be used measuring about 10 cm. 
in width by 40 cm. in length and 3 to 5 mm. in thickness; this is heated 
by a Bunsen burner or a small coal-oil stove. After the plate has a 
fairly constant temperature, the desired degree is ascertained by a 
series of drops of water, toluol (boiling point, 110° to 112° C), or 
xylol (137° to 140° C), etc., noting the line at which ebullition occurs. 
If the distance of the plate from the flame and the size of the flame, 
etc., are constant, the apparatus requires practically no attention 
and serves its purpose very well. As a rule a brief fixation only is 
necessary — i. e., exposure to a temperature of from 100° to 126° C. 
for one-half to two minutes, while in special cases Ehrlich recom- 
mends a more prolonged exposure or a higher temperature. Very 
good results are obtained for most purposes by heating the blood 
films to a temperature of 140° C. for thirty to forty-five seconds, 
as suggested by Rubinstein. This point is conveniently ascertained 
on the copper plate by noting the line at which the so-called Leiden- 
frost phenomenon begins to occur, viz., the point at which a drop of 
water assumes the spherical form and rolls about on the plate. 

In the place of the copper plate an ordinary drying oven provided 
with a thermostat and thermometer or a so-called Victor Mayer 
Siedekessel may also be employed. The latter is a small copper 
kettle covered with a thin plate, which is perforated for the recep- 
tion of the boiling tube. If a small quantity of toluol is boiled 
in this kettle for a few minutes, the copper plate will acquire a tem- 
perature of from 107° to 110° C, and retains this sufficiently long 
for ordinary purposes (Ehrlich). 

Absolute alcohol or a mixture of equal parts of absolute alcohol 
and ether (Nikiforoff) have also been recommended as fixing agents 
for blood films, but are not very satisfactory for the study of the 
neutrophilic granulation. With Ehrlich's triacid stain especially it 
will frequently be noted that the granules are stained imperfectly or 
not at all. For the study of nuclear structures, however, both are 
quite satisfactory. In the case of absolute alcohol alone immersion 
of the blood films for a few minutes is sufficient; with alcohol and 
ether fixation for one-half to two hours is necessary. 

Formalin is useful as a fixing agent and may be used in con- 
nection with practically all the common blood stains. A 1 per 
cent, alcoholic solution is employed. This is prepared by diluting 
one part of the commercial formalin, which is a 40 per cent, solu- 
tion of formaldehyde gas, with nine times its volume of water, and 
one part of the resulting solution with nine times its volume of 
alcohol. Fixation is completed in one minute, and for practical 
purposes it is merely necessary to cover the blood films with a few 



MICROSCOPIC EXAMINATION OF THE BLOOD 125 

drops of the solution, which is then drained off and replaced with 
the staining reagent directly. The method is not to be recommended, 
however, for routine purposes, as it interferes with various stains and 
often changes the normal chromatophilia. The same may be said of 
the use of concentrated solutions of bichloride of mercury, which also 
is useful for some purposes, but not for routine work. 

If the dyes are used in alcoholic solutions, as with Jenner's stain, 
Hasting's, Wright's, or Giemsa's stain, no previous fixation is necessary. 

The Aniline Dyes and the Principles of Staining.— The aniline 
dyes with which we have to deal in the clinical laboratory are all 
derivatives of hydrocarbons and all contain the benzol ring. Their 
staining properties are dependent upon the presence in the individual 
compounds of two distinct atomic complexes which are spoken of as 
chromophoric and auxochromic groups, respectively. The presence 
of the chromophoric group imparts chromogenic properties to the 
substance, the dye itself resulting on the further introduction of an 
auxochromic group. The auxochromic groups are salt-forming 
radicles and render the dye either basic or acid. Two markedly 
auxochromic radicles are known, viz., the strongly basic amino group 
— NH 2 and the feebly acid hydroxyl group — OH. Still other salt- 
forming radicles may enter into the composition of the dye, but it 
is noteworthy that these have but feebly developed auxochromic 
properties. Radicles of this order are notably the carboxyl group 
— COOH, the sulphoxyl group — S0 2 OH, the nitro group — N0 2 , and 
the nitroso group — NO (which two latter may also occur as chromo- 
phoric radicles). As the chromophoric radicle itself may have acid 
or basic tendencies it is manifest that the ultimate reaction of the 
individual compound will depend upon the inter-relation of the sum 
of its acid and basic radicles. Markedly acid dyes will result if both 
the chromophoric group and the salt-forming radicles are acid, 
while strongly basic dyes will be the outcome if both have basic 
tendencies. Between these two extremes various possibilities exist, 
the ultimate reaction depending upon the character of the chromo- 
phore, the presence of acid or basic salt-forming radicles, the simul- 
taneous presence of both, their number, etc. We may accordingly 
divide the various dyes into the following classes: 

1. Basic amino dyes. 

2. Acid nitroso dyes. 

3. Acid sulpho- and nitro dyes, viz., amino- or oxysulphonic acids, 
aminooxysulphonic acids, nitrophenols, nitroamins, nitroaminosulpho 
acids, nitrooxysulpho acids, nitroaminooxysulpho acids. 

4. Acid oxy- and oxycarbonic dyes. 

5. Aminooxy-, aminocarbonic, and aminooxycarbonic dyes. 

6. Aminosulphocarbonic-, oxysulphocarbonic-, aminooxysulpho- 
carbonic-, aminonitrocarbonic-, oxynitrocarbonic-, aminooxynitro- 
carbonic-, and aminooxysulphonitrocarbonic dyes. . 



126 THE BLOOD 

Of chromophoric groups, some twenty are known, and it is cus- 
tomary to classify the aniline dyes on the basis of these underlying 
radicles. A common characteristic of all chromophoric groups is 
the fact that they impart a quinone-like structure to the respective 
compounds. We find: 

The — N0 2 group in the nitro dyes (picric acid, Martius yellow, 
naphtol-yellow S, aurantia). 

The — NO group in the nitroso dyes (Echtgrun, naphtol green). 

— N=N — in the azo dyes (aniline yellow, chrysoidin, vesuvin, 
Sudan G and III, alizarine yellow FS, Ponceau, Bordeaux, amaranth, 
coccinin, orange G, tropeolin, Biebrich scarlet, congo, benzopur- 
purin) : 

/ in the rosanilins (malachite green, brilliant green, methyl 

C^T II violet, methyl green, fuchsin, acid fuchsin, iodine 
~~ I green, aniline blue, alkali blue, water blue, aldehyde 
green). 

Cr- in the rosolic acid dyes (aurins). 

X R— O 

C^ in the phthaleins (eosin, spriteosin, erythrosin, phloxin, 

R— CO r ose bengale, rhodamin, gallein, cerulein). 

I — O ' 

/ C0 \ in the anthraquinones (alizarin, purpurin, anthragallol, 
\nr } / alizarin blue). 

N / in the indamins (phenylene blue, Bindschedler's green, 

| \r— N toluylene blue). 



I \r_o in the indophenols (indophenol blue). 

I _l 

/^\ 
^\ / S in the thiazins (Lauth's dyes); (Lauth's violet or thionin, 

methylene blue, methylene red, methylene green). 

~^= 

— N— in the azins (eurhodin, eurhodol, toluylene red, the safranins, 

_N— Magdala red, mauvein). 

/ R \ C0 in euxanthinic acid and possibly in galloflavin (jaune 
\r/ indienne). 

in the quinolins and acridins (cyanin, quinolin red, quinolin 
%/ yellow, acridin red, and scarlet). 

N 



MICROSCOPIC EXAMINATION OF THE BLOOD 127 

The majority of the aniline dyes are found in the market in the 
form of salts of the respective staining acids and bases, and it is 
noteworthy that the latter as such are for the most part either 
colorless or but feebly stained. Triaminotriphenylcarbinol is thus 
colorless, while its monacid salts are red (fuchsin); phenolphthalein 
likewise is colorless, but forms red salts with the alkalies; fluorescein 
is pale yellow, but forms the bright-red, fluorescent uranin with 
alkali, etc. The phenols and nitrophenols, however, are commonly 
used as free acids. 

During the process of staining the salts of the staining acids or 
bases are probably decomposed by the animal or vegetable tissue 
and new compounds result between the free staining acid or base and 
the various chemical components of the tissue in accordance with the 
reaction of its component parts. The acid nuclear substance of cells 
thus shows a special affinity for basic dyes, and the basic proto- 
plasm for acid dyes. Contrasted with this chemical process of 
staining is the physical process in which the dye is merely stored in 
the pores of the tissue. Both must be sharply differentiated the one 
from the other in attempting to draw inferences in reference to 
chemical affinity on the part of component parts of a tissue or a cell. 

While in former years simple dyes were commonly employed in 
the clinical laboratory and tissues were stained successively if more 
than one dye was used, it has. been shown that it is possible to com- 
bine acid dyes with basic dyes in such manner that the acid and 
basic affinities become more or less completely satisfied. The 
resulting compounds are spoken of as neutral dyes. In these the 
staining principles of the original components are preserved and in 
addition such compounds may show new staining properties which 
are dependent upon the union of the component dyes. They are 
accordingly termed 'polychrome dyes. 

The credit of having first prepared such neutral dyes belongs 
to Ehrlich, whose triacid stain was for many years used almost exclu- 
sively in the clinical laboratory. 

A well-known representative of the so-called neutral dyes is the 
eosinate of methylene blue. Eosin is a dibasic acid and can be 
represented by the formula 

.OK 

Eo( 

^COOK 

Three compounds with methylene blue thus appear possible, viz. : 

•O meth. blue /OK /O meth. blue 

Eo/ ; Eo( . Eo( 

X COOK ^ COO meth. blue ' \ COO^th. blue 

1 II III 



128 THE BLOOD 

Although the dye has not been analyzed it is thought that formula 
I or ii expresses its constitution. It would thus not be a true neutral 
dye, but a monacid salt. As a matter of fact other so-called neutral 
dyes are strictly speaking not neutral. Ehrlich's triacid stain is 
so called because it was assumed that the three basic radicles of the 
methyl green were all satisfied by the corresponding acid radicles of 
acid fuchsin and orange G. The existence of such a triacid salt is, 
however, impossible in aqueous solutions, even if it could occur 
theoretically, which in itself is impossible, as methyl green can only 
form triacid salts with concentrated mineral acids. 

Practically important is the fact that two solutions of neutral 
mixtures can be directly mixed if they have one component in common, 
as in the case of Ehrlich's triacid stain, where methyl green is the 
common component. 

While the simple dyes, both basic and acid, are soluble in water, 
the neutral dyes are practically insoluble, but soluble in an excess 
of either the acid or the basic component, and more especially the 
former. If then an aqueous solution of methyl green is added 
carefully to an aqueous solution of acid fuchsin, fuchsinate of 
methyl green is formed at once, but at first remains in solution 
owing to an excess of the acid dye. Upon the further addition of 
methyl green, however, a point is reached when the fuchsinate 
separates out, and if the amounts of the two components have been 
carefully determined beforehand the filtrate may be nearly colorless. 
If then an excess of methyl green is added, a certain amount of the 
fuchsinate will redissolve; and if the excess be sufficiently great, the 
entire precipitate will pass into solution. 

Aside from an excess of the acid or basic component of the neu- 
tral dye its solution can also be brought about in other ways, as with 
alcohol (notably methyl alcohol), acetone, methylal, etc. 

Not all simple dyes are equally well adapted for the preparation 
of neutral dyes. Of basic dyes, the most useful are those which 
contain the so-called ammonium group, notably methyl green, 
methylene blue, amethyst blue, and to a certain extent also pyronin 
and rhodamin; of acid dyes, the readily soluble salts of the polysul- 
phonic acids, such as orange G, acid fuchsin, and narcein, and of 
the salts of the carbonic acid eosin. 

Neutral mixtures may then be prepared which contain two or 
more component dyes. If it is desired to prepare a tricolor mixture 
two possibilities suggest themselves, viz., a mixture containing one 
acid dye and two basic dyes, or one with one basic dye and two acid 
dyes. 

The principle of staining with neutral dyes is the same as in the 
case of the simple acid or basic dyes. Taking the leukocytes, for 
example, the nucleins will be found to decompose the neutral body 
and to unite with the basic component; the eosinophilic granules 






MICROSCOPIC EXAMINATION OF THE BLOOD 129 

similarly decompose the dye, but take up the acid component, while 
in the case of the neutrophilic granules we may imagine that no 
decomposition is effected, but that the neutrophilic material unites 
directly with the neutral molecule. 

Of the large number of staining mixtures which have been intro- 
duced within recent years, and of which many are mere modifications 
the one of another, I have placed only a small number of the more 
common ones before the reader at this place, and those only which 
personal experience has taught me to be useful and reliable. Where 
special mixtures are required in special work, they will be found 
described in their proper connection. 

For routine work I should suggest Jenner's method or one of the 
Romanowsky modifications as described below, notably Giemsa and 
Hastings and Goldhorn. Ehrlich's triacid stain is retained in this 
edition because it is still used as a routine stain in some laboratories. 
It is largely of historical interest, however, and less valuable than the 
others which are mentioned. 



Methods of Staining. 

General Methods. The Eosinate of Methylene Blue (Jenner). 1 — 
Equal parts of a 1.2 to 1.25 per cent, aqueous solution of eosin and a 
1 per cent, aqueous solution of methylene blue are mixed in an open 
basin and allowed to stand for twenty-four hours. The resulting pre- 
cipitate — the eosinate of methylene blue — is washed with water, col- 
lected on a filter, dried at a moderate temperature, and finely powdered. 2 
The dye can then be stored in bottles and is perfectly stable. For 
staining purposes a 0.5 per cent, solution in absolute methyl alcohol 
is employed; this can be used at once and keeps indefinitely. I have 
used this stain as a routine stain for years and can speak definitely of 
its value. 

1 Lancet, 1899, vol. i, p. 370. Simon, Maryland Med. Jour., April, 1900. 

2 The reaction probably takes place according to the formula : 

C^C:iir::oi>° (CH 3 ) 2 N— C 6 H 3 
^C 6 H ' +H 2 0+N( )S 

^CO X C 6 H 3 ^— N (CH 3 ) 2 ~ 



— o 1 

/C 6 HBr 2 .OH 
C^-C 6 HBr 2 .OH 



\C1 



>0 



C 6 H 4 — COOH 

O. NH (CH 3 ) 2 — /CsH, 



N (CH 3 ) 2 
X C1 



130 THE BLOOD 

In preparing the dye I first weigh out the requisite amount of 
eosin and methylene blue. The eosin is placed in a mortar or evap- 
orating dish and rubbed into a paste with a small amount of water; 
more water is then added until all the dye is well dissolved. This 
solution is poured into a large saucepan and diluted to the proper 
point. The methylene blue is now similarly brought into solution, 
though with a little more difficulty, as the dye is inclined to be lumpy ; 
it must all be dissolved. This solution is poured directly into the 
eosin solution and the requisite amount of water further added. 
The mixture is stirred with a rod and left to stand for twenty-four 
hours. 

If the proper quantities have been used and well dissolved, 
the filtrate is but little colored, in which case not much washing is 
necessary; if, however, there is a distinct excess of either dye this 
must be washed out The precipitate is dried at a temperature not 
exceeding 60° C, and is then powdered. The alcoholic solution 
finally is prepared by rubbing up the dye with the alcohol in a por- 
celain dish. Absolute methyl alcohol must be used. 

The blood films (on slides), which must be prepared without any 
pressure (the spreading slide should really/ be in contact only with the 
blood and not with the underlying slide), are not fixed before staining; 
this is accomplished by the absolute alcohol during the staining. The 
specimens are well covered with the stain and after about three 
minutes washed off with water and dried in the air. Care should be 
had during the staining that the preparations are thoroughly covered 
with the dye, as otherwise some of the stain is apt to become precipi- 
tated as the result of evaporation. After drying, the specimens can 
be examined directly in a drop of cedar oil. With the precautions 
stated, and by strictly adhering to the method as described, even the 
beginner can obtain perfect results. For routine purposes I can 
recommend the stain without reserve. The differentiation is ex- 
cellent and most extensive (see Plates III, IV, and VI). The red 
corpuscles are stained a grayish terra cotta, the nuclei of the leuko- 
cytes and nucleated red cells blue, the plaques mauve, the neutro- 
philic granules a purplish red, the eosinophilic granules bright red, 
and the mast-cell granules dark violet. Granular degeneration and 
polychromasia of the red cells is well shown (Plate III). Malarial 
organisms, bacteria, and filarias are stained blue. 

The May-Griinwald stain, which is frequently referred to in the 
German literature, is essentially the same as Jenner's. 1 

Ehrlich's Triacid Stain. 2 — The preparation of a reliable triacid 
stain, according to Ehrlich, presupposes the use of chemically 
pure dyes, such as those prepared by the Actiengesellschaft fur 

1 Centralbl. f. inn. Med., 1902, No. 11, and Deutsch. Arch., vol. lxxix, Heft 
5 und 6. 

2 Ehrlich-Lazarus, Die Anaemie, loc. cit. 



MICE OSCOPIC EX A MINA TIOX OF THE BLOOD 131 

Anilinfarbstoffe of Berlin. Saturated aqueous solutions of orange G, 
acid fuchsin, and methyl green are first prepared and allowed to 
clear by standing for at least one week. It is essential that these 
solutions should be perfectly clear, and it is well in measuring off 
the requisite quantities to remove the supernatant portion with a 
pipette. 

The various components are then mixed in a clean bottle, making 
use of the same measuring glass, and without washing between 
the addition of the individual components. These are taken in 
succession as shown below, and after adding the methyl green the 
mixture is thoroughly stirred until the remaining portion of alcohol 
and glycerin has been added. 

Orange G solution 13.0-14.0 c.c. 

Acid fuchsin solution 6.0-7.0 c.c. 

Distilled water 15.0 c.c. 

Absolute alcohol 15.0 c.c 

Methyl-green solution . 12.5 c.c. 

Absolute alcohol . . •. . . . . ' . . . 10.0 c.c. 

Glycerin 10.0 c.c. 

The solution is ready for use at once and does not deteriorate 
with age. 

In order to obtain the best results, it is practically necessary to fix 
the blood films by heat; fixation by Xikiforoff's method does not 
furnish constant results, and only too often leaves the neutrophilic 
granules unstained or imperfectly stained. Fixation at a high tem- 
perature (140° C), as suggested by Rubinstein, furnishes better 
results than the lower temperatures originally advised by Ehrlich, 
as the difference in color between the neutrophilic granules and the 
eosinophilic granules is brought out more prominently. The blood 
specimens are stained about five minutes, then washed in water, 
dried (by blotting, if desired), and examined as usual. 

In properly stained specimens the eosinophilic granules present a 
copper or a yellowish-red color, while the neutrophilic granules are 
violet. The mast-cell granules remain colorless and appear as round 
vacuoles in the faintly bluish-green protoplasm. The nuclei of the 
leukocytes present a greenish color and are not well stained. The 
red cells in properly heated specimens are orange; if the tempera- 
ture was too high they are yellow, and it will be found that their 
structure has suffered as a consequence. If the temperature has been 
too low the red cells take on the fuchsin. The nuclei of the nor- 
moblasts are intensely stained; the older nuclei appear black; mega- 
loblastic nuclei, on the other hand, are rather feebly stained, and in 
some specimens, indeed, the inexperienced will at first sight not 
discern any nucleus. Granular degeneration is not shown and 
polychromatophilia cannot be well demonstrated. Malarial organ- 
isms are imperfectly shown. The differentiation with the triacid 



132 THE BLOOD 

is thus markedly less than in the case of the eosinate. This is owing 
to the peculiar character of the methyl green, which is a specific 
nuclear dye. To counteract some of these deficiencies, Ehrlich has 
suggested to stain the preparations for a few seconds with an aqueous 
solution of methylene blue first, and to stain with the triacid after- 
ward. This improves the pictures somewhat, but it is not wholly 
satisfactory. 

The Romanowsky Method. 1 — The history of the Romanowsky 
method is intimately associated with the study of the minute structure 
of the malarial organism, in which the presence of a nucleus was 
first demonstrated by its aid. The dye is essentially an eosin-methy- 
lene-blue mixture, the specific staining action of which is, however, 
not due to the methylene blue per se, but to a decomposition product 
of the methylene blue, viz., methylene azure. 2 This apparently 
combines with eosin to form a neutral dye analogous to the eosinate 
of methylene blue, and can similarly be used as a routine blood stain 
in the clinical laboratory. As a rule we do not employ solutions of 
the pure dye, however, but solutions of methylene blue containing a 
variable amount of the methylene azure, to which the requisite 
amount of eosin is added. 

The following modifications of the original Romanowsky method 
are based in principle upon the above considerations: 

Hastings' Method. 3 — Three solutions are prepared, viz., (1) a 1 per 
cent, aqueous solution of eosin (Griibler's water soluble, yellow shade) ; 
(2) a 1 per cent, aqueous solution of methylene blue (Ehrlich's recti- 
fied), and (3) a solution of polychrome methylene blue. 

The polychrome methylene-blue solution is made according to 
the formula: methylene blue (Ehrlich's rectified) 2 grams, sodium 
carbonate (dry powder) 2 grams, distilled water 200 c.c. The carbo- 
nate is dissolved in hot distilled water and the methylene blue rubbed 
up in the proportion indicated. The solution is boiled over a free 
flame or kept on a boiling water bath for ten to fifteen minutes; 30 
to 40 c.c. of water are added for each 100 c.c. to allow for evaporation. 
The boiling is continued for ten to fifteen minutes longer. The hot 
solution is poured off from the sediment, and if necessary brought to 
the 200 c.c. mark by diluting with distilled water, after which it is 
partially neutralized with dilute acetic acid (12.5 to 20 per cent. 

1 St. Petersburg, med. Woch., 1891, and Nocht, Encyk. d. mik. Tech., vol. ii, 
Urban u. Schwarzenberg, Berlin-Wien, 1903, p 785. 

2 Methylene azure is an amphoteric dye, i. e., a dye of basic constitution with 
acid properties; it is the sulphone of methylene blue and has the formulas: 

7 C 6 H 3 - N (CH 3 ) 2 

n( )so 2 

1 N (CH 3 ) 2 

\ci 

3 Jour. Exper. Med., vol. vii, p. 265. 



MICROSCOPIC EXAMINATION OF THE BLOOD 133 

solution). Hastings points out that it is well to add the acetic acid 
to one-half of the polychrome-blue solution until a well-marked acid 
reaction to litmus paper is obtained (6 or 7 c.c. of 12.5 per cent, acid, 
or 3 or 4 c.c. of the 20 per cent, acid to 100 c.c.) and to mix this neutral- 
ized portion with the other half, so as to prevent ove neutralization. 
The solution should be alkaline in final reaction, since a slight excess 
of acid destroys the polychrome properties, which cannot be restored 
by the addition of alkalies. 

The three solutions are then mixed in the following proportion and 
in the following order: 

DistiUed water 1000 c.c. 

1 per cent, eosin solution 100 c.c. 

Poh T chrome-blue solution 200 c.c. 

1 per cent, methylene-blue solution 70 c.c. 

The mixture is stirred. A green, metallic-looking scum appears 
on the surface and a fine precipitate separates out. To bring this 
about it may be necessary' to add a little more of the 1 per cent, 
methylene-blue solution, viz., 80 instead of 70 c.c. 

The mixture may be filtered at once or after standing for twenty 
to thirty minutes. The residue is allowed to dry in the air or in the 
drying oven at a temperature not above 60° C. It is finally pulverized 
and can be stored in this form. The amounts of the dyes indicated 
above furnish from 0.7 to 1 gram of the ultimate product. 

For staining purposes a 0.25 per cent, solution in absolute methyl 
alcohol is used, which is prepared by rubbing up the dye with the 
alcohol in a mortar. If successful the solution has a purple plum 
color. 

Care should be had that the alcohol is neutral. Some lots of methyl 
alcohol show an acidity of 1 to 2 c.c. of T \ alkali for 100 c.c. 
Such specimens must be neutralized by the addition of 0.05 to 0.1 
gram of dry sodium carbonate for 100 c.c. 

Previous fixation of the blood specimens is not necessary, as the 
alcohol fixes while the staining is going on. The films are covered 
with the solution and left for one minute, after which they are differ- 
entiated by the addition of water until a greenish, metallic-looking 
scum appears on the surface (15 drops to a slide). This is continued 
for five minutes, when the preparations are rinsed for two to three 
seconds in water and immediately dried by blotting. This procedure 
will answer for all ordinary purposes, and for bringing out the young 
forms of the malarial parasite, but for the maturer forms it is better 
to stain for two minutes and to differentiate for ten. 

The negative surface of the specimen should be carefully inspected 
and washed if necessary, to remove any dried stain that may be present 
and which appears as a thick, greenish coating. 

In a properly stained specimen the red cells appear red; in over- 



134 THE BLOOD 

stained or old specimens light gray or light blue. Polychromatophilia 
and granular degeneration are well shown. The neutrophilic gran- 
ules are bright red, the eosinophilic granules eosin colored, and the 
mast-cell granules dark red. The nuclei of the lymphocytes, large 
mononuclear leukocytes, and myelocytes are magenta red; those of 
the polynuclear leukocytes a bluish violet. In some of the lympho- 
cytes and large mononuclear leukocytes Michaelis' granules will be 
seen. The blood plates are pale blue with red nuclei. The nuclei of 
the red blood corpuscles are red. The malarial organisms present a 
blue body with one or more intensely red nuclear structures, varying 
in size from that of a tiny dot in the youngest forms to a structure which 
in the microgametocytes fills the entire body of the parasite in the 
form of a fine reticulum. In the segmenting bodies each segment 
contains a red nucleus, while the body is blue. 

Leishman's Method. 1 — Leishman also makes use of the isolated 
eosinate of methylene blue mixed with eosinate of methylene azure. 
He proceeds as follows: Two solutions are prepared: one a 1 pro 
mille solution of eosin (Griibler's extra B. A.) in distilled water; the 
other a 1 per cent, solution of medicinal methylene blue (Griibler), 
also in distilled water, and alkalinized with sodium carbonate to the 
extent of 0.5 per cent. This last solution is heated to 65° C. for 
twelve hours, and is then allowed to stand at the temperature of the 
room for ten days before using. Equal volumes of the two solutions 
are mixed in a large open basin and allowed to stand for from six to 
twelve hours, the mixture being stirred from time to time with a glass 
rod. The resulting precipitate is collected on a filter, thoroughly 
washed with distilled water, dried, and powdered. A 0.15 per cent, 
solution of the dye in pure methyl alcohol serves as stain and does not 
deteriorate on keeping. Special fixation is not required. The 
blood film is covered with the solution and stained for about one- 
half minute. Double the amount of distilled water is then added and 
allowed to mix with the alcoholic solution. After five to ten minutes 
the stain is washed off with distilled water, a few drops of water being 
allowed to rest on the film for a minute. The specimen is next dried 
(without heat) and can be examined as usual. The soaking in water 
for a minute after the staining is important, as it intensifies the Roman- 
owsky stain; it changes the tint of the red corpuscles from a greenish- 
blue to a transparent pink or greenish color, while the nuclei of the 
leukocytes are usually a ruby red. The nuclei of nucleated red cells 
are almost black and the extranu clear portion gray. The blood plates 
are a deep ruby red with shaggy margins, frequently showing a pale- 
blue peripheral zone surrounding the red centre. The body of the 
malarial parasite stains blue and its chromatin a ruby red. In the 
case of the tertian parasite Schuffner's dots are well marked in the 
containing red corpuscles. 

1 Brit. Med. Jour., September 21, 1901. 



MICROSCOPIC EXAMINATION OF THE BLOOD 135 

Wright's Modification of Leishman's Method. 1 — Wright 
has simplified Leishman's method in several important particulars, 
which render it even more convenient for routine work; he has ascer- 
tained, moreover, that any one of the Gnibler methylene blues can 
be employed for the purpose of obtaining a sufficient quantity of 
methylene azure. 

The staining fluid is prepared as follows : 1 per cent, of methylene 
blue is added to a 1 per cent, aqueous solution of sodium bicarbonate, 
when the mixture is steamed in an Arnold steam sterilizer for one 
hour. On cooling, the solution is poured directly into a large dish or 
flask and treated, while stirring or shaking, with a sufficient quantity 
of a 1 pro mille solution of eosin (yellow shade) until the mixture 
has assumed a purple color and a scum with a metallic lustre forms 
on the surface. This will require about 500 c.c. of the eosin solution 
for 100 c.c. of the methylene-blue solution. The resultant precipitate, 
which contains both eosinate of methylene blue and eosinate of 
methylene azure, is collected on a filter, and without further washing 
allowed to dry. When thoroughly dry, a 0.3 per cent, solution in 
'pure methyl alcohol is prepared (this is practically a saturated solu- 
tion). The solution is filtered and to the filtrate 25 per cent, methyl 
alcohol further added so as to dilute the stain somewhat and to lessen 
the tendency of the dye to become precipitated during the process of 
staining. 

The air-dry blood films are covered with the stain for one minute; 
water is then added drop by drop until the staining fluid becomes 
semitranslucent and a reddish tint becomes visible at the margins, 
while a scum with a metallic lustre forms on the surface. The amount 
of water required will vary with the amount of staining fluid on the 
preparation, but in general it may be said that 8 or 10 drops will 
suffice if a seven-eighths inch square cover-glass is used. The staining 
fluid, thus diluted, is allowed to remain on the preparation for two or 
three minutes, during which time the real staining of the specimen 
takes place. It is then washed off, when the blood film will be seen 
to have a blue or purple color. 

The next step is to develop the differential staining of the various 
elements in the preparation. This is done by washing the prepara- 
tion in water, preferably distilled water, until the better-spread por- 
tions of the film appear yellowish or reddish in color. If desired, 
the process of differentiation may be readily observed by placing the 
cover-glass, film side uppermost, on a slide, covering it with water, 
and examining it with the microscope under a low magnifying power. 
The red blood corpuscles, which, as before stated, at first have a blue 
color, will become greenish, then yellowish, and finally orange or 
pinkish in color, depending upon the depth of the original staining, 

1 Jour. Med. Research, 1902, vol. vii. 



136 THE BLOOD 

which varies with the length of time that the diluted staining fluid 
has been allowed to act, and with the degree of its dilution. 

The differentiation by washing in water seems to be essentially a 
process of decolorization by which some of the blue constituent of 
the dye is removed, for the water that drains off from the preparation 
has a blue color. This differentiation or decolorization proceeds 
slowly, and may require from one to three minutes, depending upon 
the intensity of the staining and upon the tint sought to be obtained 
in the red corpuscles. 

It is apparent from the above that with a little experience with 
the method the color of the red corpuscles may be made either orange 
or pink. When the desired color is obtained in the red corpuscles 
the preparation is quickly dried between layers of filter paper and 
mounted in balsam. It is important to arrest the decolorization by 
drying the preparation as soon as the desired tint in the red corpsucles 
is obtained, for it may be carried too far. 

Dried blood films may be kept for weeks without impairment of 
their staining properties. Films months old will probably not give 
good results. 

In a suitably stained specimen the red cells are either orange or 
pink; polychromatophilia and granular degeneration are well shown 
(the granules blue); the neutrophilic granules are a reddish lilac; 
the eosinophilic granules eosin colored; the mast-cell granules a dark 
blue, a dark purple, or even black. The lymphocytes have dark 
purplish-blue nuclei with robin's egg blue protoplasm, in which the 
granules described by Michaelis appear dark blue or purplish. The 
large mononuclear leukocytes present a blue or dark lilac colored 
nucleus, and in some Michaelis' granules can also be made out. 
The blood plates are stained a deep blue or purplish and the malarial 
organisms are colored as with Leishman's method. 

Giemsa's Method. 1 — Giemsa's stain has the following composition: 

Azure II (azure plus methylene blue aa) 3.0 

Eosin (B. A.) 0.8 

Glycerin (Merck, C. P.) 250.0 

Methyl alcohol (Kahlbaum I) 250.0 

It is prepared by grinding up the dyes in the absolute alcohol and 
then adding the glycerin. The blood films are fixed for a minute 
in absolute methyl alcohol and then stained for five minutes in a 
mixture of 14 drops of the dye to 10 c.c. of distilled water, 
which is always freshly prepared; a trace of sodium carbonate may 
be added to the water to intensify the basic colors. After washing 
in water the films are blotted and are then ready for examination. 
The various elements are stained as with the methods already 
described. 

1 Centralbl. f. Bakter. Abt. I, vol. xxxvii, 2, p. 308 



ENUMERATION OF THE CORPUSCLES OF THE BLOOD 137 

Goldhorn's Method. 1 — The blood smears are fixed with pure methyl 
alcohol for fifteen seconds, washed in running water, stained for 
thirty seconds in a 1 per cent, aqueous solution of eosin, washed, 
stained for one minute in Goldhorn's polychrome methylene blue, 
again washed and dried in the air. 

The polychrome methylene blue is prepared as follows: 2 grams of 
methylene blue and 4 grams of lithium carbonate are dissolved in 300 
c.c. of warm water. The solution is heated in a porcelain dish on a 
boiling water bath for 15 minutes, then poured into a glass-stoppered 
bottle and set aside for several days. Finally it is rendered only 
slightly alkaline by the careful addition of 4 to 5 per cent, acetic acid 
solution (test with litmus paper). The method gives excellent results. 



DEMONSTRATION OF IODOPHILIA. 

Cover-glass specimens are prepared as usual; after drying in the 
air they are placed in a small jar containing a few crystals of iodine. 
After several minutes the films assume a dark-brown color, when they 
are mounted in a drop of a saturated solution of levulose and examined 
with an oil-immersion lens. The red corpuscles are stained light 
yellow, while the leukocytes are almost colorless. All glycogen 
granules, whether contained in leukocytes or free in the blood, are 
stained a distinct mahogany. 

This method furnishes better results than the older method of 
staining with a solution composed of 1 gram of iodine and 3 grams 
of potassium iodide in 100 grams of a concentrated solution of muci- 
lage (1 part of LugoPs solution to 100 parts of a thick mucilage. 2 



ENUMERATION OF THE CORPUSCLES OF THE BLOOD. 

Method of Thoma (Author's Modification) . 3 — The instrument con- 
sists of two diluting pipettes and a counting chamber (Fig. 19). The 
latter is ruled into 100 large squares (A, A, A), each occupying an area 
of -fa sq. mm. (Fig. 20). They are separated from one another by 
double guiding lines (a 6, a b) with an intervening distance of -fa mm. 
Where the horizontal and vertical lines intersect small squares (a, a, a) 
result, 100 in number, which accordingly have an area of ^-j-^- sq. mm. 
each. The large squares are thus bounded by rectangles (b, b,b), 
measuring 2V mm - i n width by fa mm. in length, representing an area 
of yj-jj- sq. mm. 

As the little platform (/) carrying the ruling is exactly fa mm. 

1 The New York Univ. Bull, of Med. Sci., 1901, vol. i, No. 2. 

2 Ehrlich-Lazarus, Die Anaemie, loc. cit. 

3 The counting chamber can be procured from Ernst Leitz & Co., New York. 



138 



THE BLOOD 



lower than the outside glass plate (e), each large square represents 
the base of a cube the contents of which are ^V X tV = 2T~o c ^- mm - 5 
each small square similarly corresponds to -^-q X tV = 40V0 c ^- mm -> 
and each rectangle to T -J- 7 X -jo = two CD - mm - 

1. Enumeration of the Leukocytes. — A drop of blood is procured by 
freely puncturing the finger or the lobe of the ear, after cleaning and 
drying the skin, wiping away the first drop or two, and avoiding un- 
due pressure. It is drawn into the 1 to 10 diluting pipette to the mark 




OS 



Fig 19. — Thoma-Zeiss blood-counting apparatus. A and E, red 
and white diluting pipette, respectively; B, counting chamber, seen 
from above; C, profile of counting chamber. 

1, and after carefully wiping the end is immediately 
mixed with a 1 per cent, solution of glacial acetic 
acid, containing a small amount of an aqueous 
gentian violet solution (1 c.c. for 100 c.c. of the 
dilute acid), by drawing up the mixture to the 11 
mark. The rubber tube of the pipette is detached, 
both ends of the pipette closed with the thumb 
and middle finger, and blood and diluent well 
agitated. A couple of drops are then blown out, 
A E so as to clear the capillary tube of the diluting 

fluid which has not entered the bulb of the pipette. A drop of 
the diluted blood is now placed upon the platform of the counting 
slide, and one of the cover-glasses which accompany the instrument 
adjusted in such a way as to exclude bubbles of air. The size of the 
drop should be such that, when the cover-glass is in place, it does 
not run over into the moat (g) surrounding the circular platform, nor 
even project over the sides. Turk advises that a tiny droplet of the 
pure diluting fluid be placed upon the plate D, before the diluted 
blood is placed upon the counting platform. If cover and slide 
have been previously scrupulously cleansed and slight pressure is 
now made upon the cover where it overlies the plate D, Newton's 

































































































































































































































































1 




















. 




























































There are ] 
cell 


n a 

3. 



PLATE VII. 






There are in all 144 large 3 



Turk's Counting Chamber. 



ENUMERATION OF THE CORPUSCLES OF THE BLOOD 139 

colored rings will become visible — a sign that a successful mount has 
been made. The slide is set aside for a few minutes, so that the 
corpuscles settle down, when it is examined with a high power (Leitz 
Ocular 2 and Obj. 6). 1 It will be noted that the red corpuscles are 
invisible and that the leukocytes are colored blue. For counting 
a mechanical stage is very convenient. Starting with the top row 
of large squares at the left corner, the total number of leukocytes 
in the 100 large squares is now carefully counted. This number 



ah a 


ib 




































A 




A 




A 






























a 




a 




a 
































b 




b 




b 









































































































































































































































































































































































































































































































































































































































































































































Fig. 20. — Simon's counting chamber. 

divided by 100 gives the average number of leukocytes for one large 
square. As the cubic contents of each large square are -g-g-g- cb. mm., 
it is only necessary to multiply the number of leukocytes in one square 
by 250 in order to find the number for 1 cb. mm. of diluted blood, and 
this by the degree of dilution (in the above instance by 10) to find 
the number for 1 cb. mm. of diluted blood, and 1000 X 10 the number 
in 1 cb. mm. of non-diluted blood. 



1 To focus through the thick cover the objective should have a long working 
distance. 



140 



THE BLOOD 



Example.— Total number of leukocytes counted in the 100 large 
squares = 400; hence f#£, viz., 4 = number of leukocytes in a single 
square, i. e., in ^ cb. mm. of diluted blood; hence 250 X 4 = 1000, 
the number of leukocytes in 1 cb. mm. of non-diluted blood, and 
1000 X 10 the number in cb. mm. of non-diluted blood. 

When counting the cells note should only be taken of such that 
lie within the squares or upon the upper and left boundary lines; 
cells upon the right and lower lines should be omitted. 

In the above instance a dilution of 1 to 10 has been advocated. This 
may be used as a matter of routine. If a marked grade of leukocy- 
tosis is anticipated a dilution of 1 to 20 will be found more convenient. 





Fig. 21.— Turk. 



Fig. 22. — Thoma; centre part. 












.. 




====' 











Fig. 23. — Zappert-Ewing. Fig. 24. — Thoma. 

Blood-counting chambers. 

If desired even higher dilutions may be used, in which case the red 
pipette permitting of a dilution of 1 to 100 or more is employed. 

2. Enumeration of the Red Cells. — The blood is diluted 100 times by 
filling the red pipette with blood to the mark 1 and with the diluent 
to 101. For diluting the blood in the enumeration of the red cor- 
puscles Toison's solution is most convenient: 

Sodium chloride 1.0 

Sodium sulphate 8.0 

Neutral glycerin 30.0 

Distilled water 160.0 

Methyl violet (5 B.) 0.025 



ENUMERATION OF THE CORPUSCLES OF THE BLOOD 141 

To prevent the development of molds the solution should further 
contain about 1 pro mille of thymol. 

After mixing the diluent and blood thoroughly and blowing out 
the pure diluting fluid in the capillary tube a drop is mounted as 
described. All the red corpuscles are then counted — in the 100 
small squares, if no marked degree of anemia exists, or in 40 or more 
rectangles if the corpuscles are distinctly diminished. The calculation 
is then made as follows, bearing in mind the cubic contents, corre- 
sponding to the small square and the rectangle, viz., 40 1 00 and tqVo 
cb. mm., respectively: 

Example 1. — Number of red cells in 100 small squares = 1000; in 1 
therefore 10, viz., in ^oVo CD - mm - "» m 1 CD - mm - °^ diluted blood 4000 X 
10 = 40,000 and in 1 cb. mm. of non-diluted blood 40,000 X 100 = 
4,000,000. 

Example 2. — Number of red cells in 40 rectangles = 800; in 1 
reactangle therefore *■££ = 20, i. e., in yoVo CD - mm -; m 1 cb- mm - of 
diluted blood hence 20 X 1000 = 20,000, and in 1 cb. mm. of non- 
diluted blood 20,000 X 100 = 2,000,000. 

If for any reason a larger area is to be counted for red cells, this 
can, of course, be readily done by going over a larger number of rect- 
angles, or by combining small squares and rectangles, due allowance 
being made for the cubic contents of the ground covered. 

Other counting chambers are also in existence. The form of the 
ruling of various models is shown in the accompanying figure (Figs. 
21 to 24). They are used in the same manner as that of the author. 
The calculation in each case depends upon the number of squares 
counted, and its corresponding cubic contents and the degree of 
dilution. 

Second Method. — If a counting chamber with one of the more 
modern rulings is not available, but if a mechanical stage is at hand, 
the leukocytes can also be counted with the old Thoma counter 
in the following manner: A drop of the diluted blood is mounted as 
usual. With the mechanical stage a field corresponding to the 
position of 1 in the accompanying diagram (Fig. 25) is then selected 
as the starting point. The presence or absence of leukocytes is noted 
and the field changed, so that an adjoining circle is brought into 
view, and so on. In this manner at least 100 circles are gone over, 
using a corpuscle to the side or above or below as a guide to the next 
field. The total number of leukocytes is noted and the average 
for one circle calculated. If the cubic contents corresponding to 
each circle are known, the calculation of the number of leukocytes 
in 1 cb. mm. of blood becomes a simple matter. The determination 
of the cubic contents corresponding to a circle is made as follows: 
Noting the number of the eyepiece and the objective, the diameter of 
the field of vision is measured with a stage micrometer, or with the 
aid of the rulings of an ordinary Thoma-Zeiss counter, bearing in 



142 



THE BLOOD 



mind in the latter case that the distance between two vertical lines 
is yV mm - The area of the circle, according to geometrical law, will 
then be equivalent to rep 2 , in which n is a constant factor — i. e., 3.1416; 
and p the radius, from which the corresponding cubic contents are 
calculated by multiplying the result by 0.1 — i. e., the depth of the 
counting chamber. The resultant value, which should be ascertained 
for every instrument separately, will, of course, be constant for the sys- 
tem of lenses and the counting chamber used. With a Bausch & 
Lomb i (long-working distance), the 1-inch eyepiece, and 160 mm. 
tube length, the cubic contents of the field are 0.009 cb. mm. 

Example. — The blood was diluted 100 times. In 100 fields 50 
leukocytes were noted — i. e., 0.5 for 1 field, or for 0.009 cb. mm.; 
in 1 cb. mm. of diluted blood there would hence be 0.5 divided by 




Fig. 25. — Schema of circles for counting leukocytes: a, moat surrounding central 
platform, b, of counter; 1, starting point. 



0.009 = 55.5, and for 1 cb. mm. of undiluted blood 55.5 X 100 = 
5550 leukocytes. 

Cleaning of the Apparatus. — After use the apparatus must be care- 
fully cleansed. The pipette is washed out with the diluting fluid, 
then with water, next with absolute alcohol, and finally with ether. 
The washing will be facilitated by slipping the rubber tube over the 
long arm of the pipette and blowing the contents of the bulb out of 
the short arm. In laboratories which are equipped with a suction 
pump this may be conveniently employed; the entire process then 
occupies only two or three minutes. 

. The counting chamber is washed with water only; alcohol and ether 
dissolve the substance with which the platform is cemented to the slide. 



ENUMERATION OF THE CORPUSCLES OF THE BLOOD 143 

Differential Enumeration of the Leukocytes. — The differential enu- 
meration of the leukocytes is usually made in dried and stained speci- 
mens. A mechanical stage is a great convenience, but not a necessity. 
The idea is to go over a large number of cells, for ordinary purposes 
not less than 500 to 600, to classify these, and finally to calculate the 
percentages. The cells are charted as shown below: 

S. M. (small mononuclear leukocytes) :?Hl VU ML Wl 

mi m m vu mi = 45 

L„ M. and T. F. (large mononuclear leukocytes and transition 

forms): mi TfU THl ='15 

P. (polynuclear neutrophiles) : mi THl THl Ml 7M 

mimimrmmmimimimimi 
mrmmimiMmimimimimi 
mi mi mi m mi mi =155 

E. (eosinophils) : /)^( — 5 

M. (mast-cells) : // =2 



222 



Result : Total number of cells counted, 222, of which : 

45X100 



small mono.s., 


222 — ^ *^ P er cen ^ 


large monos., 


15X100 

222 ~~ D ' 


polys., 


155X100 ." 
222 - 69 ' 8 


eosins., 


5X100 „ 
222 — Z - Z 


mast., 


2X100 

Q " 



While making a differential count it is always well to keep note 
of the time, as it is often possible in this way to form a fair idea of 
the actual number of the leukocytes without an absolute count. This, 
of course, requires a certain amount of experience in the preparation 
of the smears, which should be uniformly of nearly the same thick- 
ness. After one has then learned by control how many leukocytes 
in a blood smear, observed within a certain length of time, may be 
considered as normal, it is not difficult to judge the grade of a hyper- 
leukocytosis by the increase in number noted within the same length 
of time. Everyone must here work out his personal equation. A 
general idea of the degree of increase can, .of course, be formed by 
examining the specimen with a low power — a Bausch & Lomb J , 
for example — but in the manner indicated one gets a numerical 
expression which is at times quite helpful. The count itself can be- 
readily made with a -| ; it requires a little practice, but proves a great 
saving of time. 



144 THE BLOOD 

Enumeration of the Plaques. — For this purpose the method of 
Broide and Russel has been advocated. The method is an indirect 
one. First, the red corpuscles are counted in the usual manner. 
A drop of the staining fluid, composed of equal parts of a 2 per cent, 
solution of common salt and a saturated solution of dahlia in glycerin, 
is then placed upon the finger, when this is punctured through the 
drop and the blood allowed to mix with the reagent. In this mixture 
the ratio between the plaques and the red corpuscles is ascertained, 
and the total number of plaques contained in 1 cb. mm. of blood 
determined by calculation. The plaques are stained the color of 
dahlia and can readily be counted. Rapid work is essential, as the 
staining fluid soon attacks the red corpuscles. 

Other writers determine the ratio of plaques to red cells in smears 
and then calculate their number after an absolute red-cell count. 
Jenner's stain or any one of the methylene-azure mixtures (Hastings', 
Giemsa, Wright) will answer the purpose. 

The Hematocrit. — The use of the hematocrit for counting the 
red blood corpuscles has been repeatedly advocated, but has not met 
with favor. The method is inapplicable whenever there is any 
material variation in the size and form of the red corpuscles and when- 
ever the number of the leukocytes is greatly increased. This means 
that the method cannot be employed in the majority of cases in which 
we are especially interested in the blood count. If, however, it is 
desired to ascertain the volume of the red corpuscles in relation to 
the amount of plasma, the instrument will furnish satisfactory results. 
A centrifuge run by electricity is practically a necessity; in this way 
alone is it possible to maintain the proper rate and uniformity of speed. 
Hand centrifuges are, in my experience, totally inadequate, and with 
instruments driven by water power it is impossible to attain a sufficient 
rate of speed for this purpose. An apparatus like the one pictured 
in the accompanying illustration (Fig. 26) answers the purpose best. 
It is connected with the street current or with a small battery, a 
rheostat being interposed to control the current and the rate of speed. 
At the same time a speed indicator can be attached which strikes a 
bell for every 100 revolutions. For the hematocrit a speed of 8000 to 
10,000 revolutions per minute is required. 

The hematocrit which is almost exclusively used in the United 
States is that of Daland (Figs. 27, 28, 29). It consists of a metallic 
frame which carries two glass tubes measuring 50 mm. in length 
and 0.5 mm. in diameter. Each tube bears a scale ranging from 
to 100, the individual divisions of which are rendered more easily 
visible by a magnifying lens front. In the frame the outer end 
of each tube fits into a small depression, the bottom of which is cov- 
ered with thin rubber; the inner ends are held in position by springs. 
The instrument is screwed to a firm table and is oiled daily when in use. 

If the patient is directly available, undiluted blood is used. The 



ENUMERATION OF THE CORPUSCLES OF THE BLOOD 145 



finger is washed with soap and water and alcohol, as usual, and is 
freely punctured. A small rubber tube is then slipped over the 
end of one of the hematocrit • tubes, which is completely filled by 
suction. The bevelled end of the tube is quickly covered with the 
finger, which has been previously lubricated with a little vaselin; 
the rubber tube is disconnected, and the glass tube immediately fixed 
in the one compartment of the frame. Its mate is rapidly placed on 
the opposite side and the instrument rotated at a speed of from 




Fig. 26. — Imp roved electric hematocrit, with 
fender, rheostat, and speed indicator. The 
hematocrit attachment replaces the urine tubes 
seen in the^revolving armature. 



Fig. 27. — Daland's hematocrit. 



8000 to 10,000 revolutions per minute for three minutes, when the 
volume is directly read off. In normal individuals the volume of 
the red corpuscles is approximately 50 per cent., so that in a given 
case a proportionate expression of the percentage of corpuscles, as 
compared with the normal, can be obtained by multiplying the figure 
on the scale by 2. 

If the patient is not directly available, the blood is diluted with 
an equal volume of a 2.5 per cent, solution of potassium bichro- 
10 



146 



THE BLOOD 



mate, as proposed by Daland. As Ewing suggests, this can be 
done with the pipette which accompanies the Thoma-Zeiss blood 
counter. In the case of the red pipette the capillary tube is filled 
with blood to the mark 1, then a small air bubble is drawn in, fol- 
lowed by another tube length of blood. Three or four volumes 
of blood are obtained in this way and diluted at once with an equal 
quantity of the bichromate solution. In the case of the white 
pipette a single tube length of blood and the diluent is sufficient. 
Blood and diluent are thoroughly mixed, care being had not to 
include any air bubbles. In this form the blood is carried to the 
laboratory, where both tubes are filled by allowing the drops to flow 
in from the point of the pipette. To obtain the percentage volume 
the resultant figure is in this case, of course, multiplied by 4. 

In the case of normal blood it has been ascertained that 1 per 
cent, by volume, as read off from the scale, corresponds to almost 
100,000 red corpuscles per cb. mm. ; to obtain the total number of 




Fig. 29. — Daland's hematocrit tube. 

red cells per cb. mm., it is hence only necessary to add five ciphers to 
the percentage indicated on the scale. 

Example. — Undiluted blood was used; the reading on the scale 
was 45. The volume per cent, of the red corpuscles would hence 
be 90, and the number of red cells per cb. mm. 4,500,000. 

But, as I have pointed out, this calculation presupposes that the 
size and form of the red cells are practically normal, and that the 
leukocytes are not materially increased. 

With normal blood the leukocytes appear only as a narrow, indis- 
tinct, milky band at the central end of the column of red cells, which 
with a material increase of the leukocytes becomes more marked and 
reaches its greatest extent in well-marked cases of leukemia. 

Aspelin has recently suggested that with a suitable modification 
of the Daland apparatus quite accurate leukocyte counts can be ob- 
tained by centrifugation; but bearing in mind the variations in the 
size of the different leukocytes and the varying degree in which the 



ESTIMATION OF HEMOGLOBIN 147 

different forms take part in the production of the different types of 
hyperleukocytosis, it is evident at once that still less is to be antici- 
pated from the centrifugal method in this direction than in the case 
of the red cells. 

Volume Index. — The term volume index has been introduced by 
Capps to designate the relation existing between the volume of red 
cells determined by centrifugation (see above) and their number. 

If both are normal the ratio iZZZZTelh 1 (°- 99 avera 8 e of 10 
normal individuals). In 29 cases of pernicious anemia the volume 
index was high during the active stage of the disease, ranging from 
1.05 to 2.0. During periods of improvement it steadily fell, while 
in periods of decline it steadily rose. In chronic secondary anemia 
of moderate intensity normal values are the rule; in a few they are 
low. In acute secondary anemia (sepsis, hemorrhage) the index may 
be low (0.72) ; so also in chlorosis of the severer type. In a few cases 
of chronic severe secondary anemia (as in uncinariasis) Capps found 
the volume index high. Analogous results have been obtained by 
Wroth. 

Literature.— Hedin, Arch. f. ges. Phys., vol. xl, p. 360. Gartner, Wien. klin. 
Woch., 1892, No. 2. Daland, Fort. d. Med., 1891, No. 21. Aspelin, Zeit. f.. klin. 
Med., 1903, vol. xlix, p. 393. J. A. Capps, Journ. Med. Research, December, 
1903. P. Wroth, Johns Hopkins Hospital Bull. February, 1907. 



ESTIMATION OF HEMOGLOBIN. 

Hemoglobinometers. — While it is usually possible to form a 
fairly clear idea of the degree of anemia by direct inspection of the 
patient, the appearance of the mucous surfaces, etc., it is often de- 
sirable to obtain more definite information, and, above all, a numer- 
ical expression of the extent of the anemia. This is especially im- 
portant in the diagnosis of certain forms of anemia, in which the 
"color index" plays an important part — i. e., the ratio between the 
percentage of hemoglobin and the percentage of the red corpuscles, 
as compared with the normal. To this end, special instruments have 
been devised, which are termed hemoglobinometers or hemometers. 
Of the various forms which are now in the market, the hemoglobin- 
ometer of Dare is probably the best, and is rapidly replacing the old 
instrument of v. Fleischl, Avhich for many years was the standard. 
It is more exact and more convenient. Miescher's modification of 
the Fleischl instrument is possibly still more accurate, but too costly 
for general adoption. The little instrument of Gowers, in the modifica- 
tion of Sahli, when obtained from a reliable source will also furnish 
good results. Unfortunately many of those which have been placed 
on sale are worthless. Oliver's instrument has some advantages over 
the Fleischl, but none over the Dare. The Talquist method is 



148 



THE BLOOD 



warmly recommended by Cabot, and may be used to advantage in 
routine work by the general practitioner; for exact work it is insuf- 
ficient. 

Dare's Hemoglobinometer. — The essential parts of Dare's lemo- 
globinometer (Fig. 30) are an automatic pipette for collecting the 
blood (Fig. 31) and a graduated color scale (Fig. 32) to measure 
the corresponding percentage of hemoglobin. This latter reads 
from 10 to r20, the 100 mark corresponding to the color of a solution 

of 13.77 grams of hemoglobin 




m 



100 



Fig. 30. — Dare's hemoglobinometer. 



c.c. of serum. The 
various shades of color cor- 
responding to the scale are ob- 
tained by rotation of a pris- 
matic glass semicircle tinted 
with golden purple of Cassius 
(Fig. 32, E), which is secured 
to a thin white glass disk (I). 
The numerical scale is placed 
on the edge of a correspond- 
ing semicircle (H) of thick 
white glass (F). This part 
of the apparatus is enclosed in a 
dust-proof hard-rubber case, 
and is rotated from the outside 



by the aid of a rubber-covered roller which runs on the edge of the disk 
and is turned by a milled wheel at R (Fig. 30). In the rubber case 
is a little circular window through which the color of the prism 
is viewed by means of a small telescoping camera tube (Fig. 33, N), 





Fig. 31. — Automatic pipette. 



Fig. 32. — Graduated color scale. 



provided with a magnifying lens of low power. The color aperture 
represents a surface about equal to 3 per cent, of the color scale. 
Looking through the tube a corresponding window will be seen side 
by side with the one through which the color scale is visible. In front 
of this the blood pipette is secured. The essential part of this is an 
oblong plate of white glass (Fig. 31, A), into the end of which a 
depressed surface of measured depth is ground, the floor being ex- 
actly parallel to the plane surface of the glass. This depression 
forms a capillary chamber (D) when the transparent glass plate (B) 



ESTIMATION OF HEMOGLOBIN 



149 



is firmly clamped upon it by the pipette clamp C; it is filled by 
capillary attraction when either of the three free edges is touched to 
the blood drop. The pipetteijis le d in position on the stage of the 
instrument by guides which run in grooves on the lower part of the 
clamp. The plate of white glass is toward the light. 

The camera tube screws into a movable shutter (Fig. 30); when 

this is swung outward the two aper- 
tures become visible through which the 
blood and the colored scale are viewed. 

In front of the pipette a candle is 
clamped in such a position that both 
the blood and the color scale are equally 
illuminated. 

Method of Use. — As the compari- 
son of the color of the blood with that 
of the color scale should be made as 



o N 


o 1 




-1 1 


"^ 


Hhhhv 


M 


jjj^^iSi^ 


'■ ; % 


m» "l^-i 


ff^^ 


c 


m' u'I 
N 


, 



Fig. 33.— Horizontal section of 
Dare's hemoglobinometer (on a 
level with centre of comparison 
apertures): J, candle; K, white 
glass disk of color prism; L, color 
prism; M, aperture through which 
color of blood film is viewed; M', 
aperture through which the illum- 
inated color prism is viewed; N, 
camera tubo; O, transparent glass 
of pipette; P, white glass of 
pipette. 




Fig. 34. — Filling the automatic blood pipette. 



soon after filling the pipette as possible, the apparatus is prepared 
for use beforehand by screwing the camera tube into place and ad- 
justing the candle; this should be at such a level that the blue flame 
of the candle is below the color aperture, care being taken to have 
the wick of proper length (half-inch) and not charred at the tip. 
Curved or eccentric wicks should be turned so that the intensity of 
light in a vertical position is midway between the two color aper- 
tures. 

The glass plates of the pipette having been thoroughly polished and 
refastened in the clamp, the finger or ear is freely punctured as usual 
and the capillary space of the pipette filled with the blood, by hold- 
ing one of the three edges horizontally to the drop (Fig. 34). Any 
blood adhering to the flat surfaces of the glass plates is carefully 
wiped away and the pipette placed in position. The candle is 
lighted, the shutter thrown out, the camera tube focused, and the 
color of the blood (on the left) compared with the color scale (on the 
right). The two are matched by rotating the color disk by means 



150 THE BLOOD 

of the milled wheel, which should be done in an abrupt manner, 
and frequently resting the eye. To this end the shutter is dropped 
and thrown out again as the case may be. The examination need 
not be conducted in a darkened room, but it is important to turn the 
instrument toward a dark background, so as to eliminate all direct 
or reflected light. The reading is indicated by the bevelled edge of 
the rectangular opening on the side of the case; the figure immedi- 
ately beneath this represents the percentage of hemoglobin. Im- 
mediately after use, the two glass plates of the pipette are cleansed 
with water and a little acid alcohol, dried, and again replaced. Fur- 
ther details in regard to technique accompany the instrument. 

My personal experience with the instrument has been quite satis- 
factory. 

Literature. — A. Dare, Phila. Med. Jour., Sept. 22, 1900. 

Fleischl's Hemoglobinometer. — The principle underlying the v. 
Fleischl method is essentially the same as that of the Dare method; 
the color of the blood is compared with the color of a glass wedge 
stained with the golden purple of Cassius or a similar pigment, 
a scale indicating the corresponding amount of hemoglobin. With 
the Fleischl instrument, however, diluted blood is used, which is one 
of the disadvantages of the method. 

The instrument (Fig. 35) consists of the glass wedge a, to which 
a scale, b, is attached, ranging from to 120, being placed at the 
thinnest, 120 at the thickest portion of the wedge. By means of a 
rack and pinion this may be made to slide from side to side beneath 
a platform corresponding to the stage of a microscope. In the 
centre of the platform there is a circular opening into which artifi- 
cial light (daylight is not permissible) is projected from a circular 
plate of plaster of Paris mounted beneath, in the position of the 
mirror of the microscope. Into the circular opening a metallic tube, 
1.5 cm. in height, is fixed, which is closed at the bottom with a 
plate of glass and divided into two equal compartments by a metal 
partition. One compartment receives the light through the glass 
wedge — the red chamber; the other, directly from the plaster-of- 
Paris reflector — the white chamber. 

Capillary pipettes of known capacity accompany the instrument. 
This capacity is somewhat variable and is indicated on the handle 
of each, which number must correspond with that marked on the top 
screw head of the instrument. Generally speaking, the capacity of 
each pipette is such that with the blood of a perfectly normal indi- 
vidual the mixture of blood and water in the white chamber will 
correspond in color to that of the colored wedge at the mark 100 
(a 13.77 per cent, solution of hemoglobin). 

The pipette is filled by capillary attraction from a drop of blood 



ESTIMATION OF HEMOGLOBIN 



151 



obtained in the usual manner. If on trial it is found that the blood 
does not immediately run up in the tube, this is repeatedly washed 
out with water and then dried. If this is always done after the 
examination, the pipette will be in working order on the next oc- 
casion. While filling the pipette care should be had that it is not 
immersed in the blood, but only brought in contact with it. The 
two compartments of the cell having been previously partly filled with 
water, the charged - pipette is at once placed in the white chamber 
and rapidly moved to and fro until the blood is well mixed with 
the water. Any trace remaining in the pipette is carefully washed 
out with water by the aid of a medicine dropper. The contents of 




Fig. 35. — v. Fleischl's hemometer. 



the chamber are stirred with the handle of the pipette when both 
compartments are filled with water, using the same dropper, so that 
there is a convex meniscus over each. The color of the blood is 
then matched on the wedge, which should be moved by quick turns 
of the adjustment screw rather than in a gradual way, as the eye will 
otherwise be less apt to appreciate fine shades of difference. Day- 
light, as I have said, is not permissible; a candle or gas flame of 
moderate intensity placed about a foot and a half distant is best. 
The eye should be perpendicularly above the cell, and it is well to 
view the colors through a paper tube which is placed over the two 
compartments. The number facing the notch in the little well 



152 THE BLOOD 

immediately behind the cell indicates the percentage of hemoglobin. 
The readings corresponding to the middle portion of the wedge are 
apt to be more nearly correct than the lower values. For this 
reason it is well, when a preliminary examination has shown a low 
figure, to repeat the test, using two or three pipettefuls of blood 
instead of one, the result, of course, being divided by 2 or 3, as the 
case may be. On the whole, the Fleischl method furnishes results 
which are somewhat lower than those obtained with the Dare; this 
is true especially of the older models, with which a percentage of 
100 was only rarely observed. The instruments of more recent 
construction, however, are much better. Personally I regret to see 
the Fleischl apparatus supplanted by newer instruments; it was con- 
venient and neat. It has its defects, to be sure, and it is unfor- 
tunate that the Miescher modification, in which these have been elimi- 
nated, and which unquestionably gives the most accurate results, is 
still so costly that its general use is out of the question. 

Gowers' Hemoglobinometer (Sahli's Modification). — The apparatus 
(Fig. 36) consists of two glass tubes (A and B) which are of the same 
diameter. One of these (A) is closed and contains a solution of 
hematin hydrochlorate in a concentration corresponding to a 1 per 
cent, solution of normal blood. The other tube is provided with an 
ascending scale of 140 divisions, each degree corresponding to 20 
cb. mm. A capillary pipette marked at 20 cb. mm., a guarded lancet, 
a dropping bottle, and a small stand accompany the instrument. 

The finger is punctured as usual and the pipette filled to the 20 
cb. mm. mark; the blood is immediately discharged into the graduated 
tube and mixed with one-tenth normal hydrochloric acid (saturated 
with chloroform as a preservative) which has been previously filled 
in to the mark 10. When the color of the mixture has become a clear 
dark brown, water is added drop by drop, shaking after every addition, 
until the color matches that of the standard solution. The division 
on the scale ultimately reached indicates the percentage of hemoglobin. 

The examination can be conducted with natural and artificial 
light. 

The method, as I have indicated above, is satisfactory if the instru- 
ment has been obtained from a reliable source. Its low cost makes 
it especially serviceable in large clinics and for purposes of teaching 
in the clinical laboratory. But in every case it is advisable to com- 
pare its scale with a standard instrument 

Talquist's Method. — The color of the blood, in this case undiluted, 
is compared with a series of lithographed standard tints, which repre-. 
sent a scale ranging by tens from 10 to 100. The technique is very 
simple: drops of blood are received on pieces of white filter paper of 
suitable thickness which accompany the color scale, and are compared 
with the tints on the plate, using ordinary daylight. 

Accuracy is, of course, not to be expected from so crude a method, 



EST IMA TIO N OF HEMOGL OB IN 



153 



so that its use is of necessity limited. It will suffice in a very general 
way to control the result of treatment, but it is inapplicable in the 
determination of the color index. 

Estimation of Blood Iron with Jo lies' Ferro meter. — The estimation 
of the hemoglobin from the amount of blood iron, as originally sug- 
gested by Jolles, is unfortunately not possible, as it has been shown 
that constant relations between the two bodies do not exist. All the 
iron of the blood is not present in this form, nor does it all occur in 
the form of colored compounds. Jolles' method B A 

of estimating the total amount of blood iron 
deserves consideration, however, as it is a 
practical method and discloses facts which 
are of clinical interest. It is desirable that it 
should be introduced in the clinical laboratory 
as a routine method. 

The principle is the following: A small 
amount of blood is incinerated, and the re- 
maining red oxide of iron brought into solu- 
tion with a little monacid potassium sulphate. 
In this solution the iron is then estimated 
colorimetrically with an instrument which is 
constructed upon the principle of Fleischl's 
hemometer and which is termed the ferrorn- 
eter. It is made by Reichert in Vienna and 
can be readily transformed into the hemom- 
eter proper. Full directions accompany the 
apparatus. The results are expressed in rel- 
ative terms, the number 100 on the scale 
corresponding to 0.0425 per cent, by weight 
of iron. Some of the results which have been 
obtained with the clinical ferrometer are given 
below, together with the corresponding figures 
indicating the amount of hemoglobin: FlG - ^nometer hem °" 




Ferrometer Hemometer 

number. number. 

Normal 103.0 100 

Normal 92.6 105 

Normal 95.5 100 

Normal 110.0 105 

Normal 83.8 92 

Chlorosis 32.1-68.2 30-65 

Simple anemia 33.2-74.7 15-40 

Icterus 55.0 80 

Leukemia 40.7 32 

Leukemia 38.6 35 

Pseudoleukemia . • 77.24 75-80 

Severe diabetes 78.7 30 

Severe diabetes 91.4 35-40 

Parenchymatous nephritis ..... 51 7 50 



154 THE BLOOD 

These figures at once illustrate the lack of relationship which exists 
between the amount of hemoglobin and that of the blood iron as a 
whole. 

In a series of cases Jolles also examined into the presence of iron 
in the serum, by centrifugating a given volume of blood mixed with 
an 0.8 per cent, salt solution, and found that in health the serum 
contains no iron. In 3 cases of chlorosis, in 1 case of leukemia, in 1 
of neoplasm, and 1 of interstitial nephritis, negative results were like- 
wise reached. In 2 cases of severe diabetes, on the other hand, notable 
quantities were found. 

Deganello 1 has studied the relation between the amount of blood 

/Fe\ 
iron and hemoglobin ( fjj-y in different forms of secondary anemia, 

and found that this ratio remains normal, until the Hb has reached 
a certain minimum — 46 to 58 per cent. ; from this point off the value 

Fe 

— surpasses the normal the more the deeper the Hb value falls. 

Mere mechanical loss of Hb does not materially alter this value, 
however, even in cases of marked oligochromemia. When toxic 
influences are at play marked discrepancies will result. 

Mitulescu 2 comes to quite analogous conclusions. He thinks that 
the hemoglobin estimation only is required as a rule, from which the 
iron value can be calculated according to Hoppe-Seyler's formula: 

Fe = Tr^—~ • If hemolytic processes are suspected, or if albumin- 

100 

uria exists, both methods are to be employed. 

Literature. — A. Jolles. "Ferrometer," Deutsch. med. Woch., 1897, No. 10; 
ibid., 1898, No. 7. Hladik, " Untersuchungen iiber d. Eisenghalt d. Blutes 
gesunder Menschen," Wien. klin. Woch., 1898, No. 4. S. Jellineck, " Ueber 
Farbekraft und Eisengehalt d. Blutes," ibid., Nos. 33, 34. A Jolles, "Verein- 
fachtes klin. Ferrometer," Berlin, klin. Woch., 1899, No. 44, p. 965. 



KRYOSCOPIC EXAMINATION OF THE BLOOD. 

The kryoscopic examination of the blood has for its object the 
determination of the molecular concentration, and hence of the os- 
motic pressure of the blood. The method is essentially based upon 
the observations of Raoult: (a) that all solid, liquid, or gaseous 
substances when dissolved in a liquid will lower the freezing point of 
that liquid; (b) that the degree to which the freezing point is lowered 
is dependent upon the amount of substance which is present in solu- 

1 Atti del R. Istituto Veneto di Scienze lett. and arti. T. lxiii. 

2 Zeit, f. klin. Med., lxx, 344, 190. 



KBYOSCOPIC EXAMINATION OF THE BLOOD 155 

tion; and (c) that equimolecular solutions have like freezing points. 1 
It follows that the freezing point of a solution furnishes an index 
of its molecular concentration, and hence also of its osmotic pressure, 
as this has been shown by van't Hoff to be proportionate to the num- 
ber of molecules present. 

The degree to which the freezing point is lowered is designated 
by the letter A. In the case of normal blood this varies between 
—0.56 and — 0.58° C, as compared with distilled water. A further 
depression is probably always indicative of renal insufficiency; it is 
a symptom of decided value and deserves more general consideration. 
In the domain of renal surgery especially the study of kryoscopy of 
the blood is important. Of foreign investigators, Kummel more 
particularly has pointed out the value of the method in this field. 
As the result of 265 freezing-point determinations of the blood, in 
170 cases in which various operations were performed upon the kidney 
and in which a direct examination of the organ was possible, he con- 
cludes that kryoscopy furnishes the most important index of renal 
insufficiency as compared with all other modern methods. Other 
observers, such as Casper and Richter, Tinker, and others, have 
arrived at similar conclusions. To Koranyi, however, belongs the 
credit for the introduction of kryoscopy into the clinical laboratory 
and its application to the study of renal diseases. Senator, Claude 
and Balthazar, Albarran, Kovesi, Lindemann, Waldvogel, and others 
have materially contributed to establish its value as a clinical method. 

Zangemeister, 2 who has carefully studied the molecular concen- 
tration of the blood during pregnancy, the puerperal period, and in 
eclampsia, found a lessened concentration in the first instance, and 
values in the second which were still below the normal average and 
yet slightly higher than in pregnancy. In eclampsia the average 
concentration was quite normal. Similar results have been obtained 
by others, such as Futh and Kronig, 3 Szili, 4 and Lobenstine. 5 During 
pregnancy (ninth month) the latter found the average J in 12 cases 
to be — 0.51° (variations from 0.45° to 0.57°) ; the average value in 
12 puerperal women was — 0.53° (variations: —0.49° to — 0.58° C). 
He accordingly concludes that if there is retention in eclampsia it must 
be of either colloidal substances or of crystalline substances, too small 
in amount to affect the concentration of the blood. 

Schmidt" has recently studied the kryoscopic behavior of the blood 

1 Solutions are termed equimolecular when for a constant quantity of the 
solvent they contain such quantities of substance in solution that these bear the 
same ratio to each other as their molecular weights. Example: The molecular 
weight of sodium chloride is 58.5 and of sodium carbonate 106; if we dissolve 
these quantities or the same multiples of each in a constant quantity of water, 
such solutions would be equimolecular. 

2 Zeit. f. Geburts. u. Gvn., vol. i, Heft 3 

3 Centralbl. f. Gyn., 1901, No. 25. 

4 Orvosi Hetilap, 1900, No. 37. 5 Amer. Med., October 15, 1904. 
6 Jour. Amer. Med. Assoc, Sept. 23, 1905. 



156 



THE BLOOD 



i 



in pneumonia and as the result of an analysis of 24 cases he con- 
cludes as follows: 

There is an absolute lowering of the freezing point in pneumonia, 
which seems to depend either on the extent of the consolidation or on 
the height of the temperature or both. The concentration of the 
blood increases, as shown by the lower freezing point, as the disease 
progresses up to the time of the crisis. In those cases where the heart 
weakens perceptibly the freezing point of 
the blood becomes lower and in the fatal 
cases in which the heart gives out the freez- 
ing point is very low. 

Method. — In the clinical laboratory a 
modification of Beckmann's apparatus is 
most conveniently employed (Fig. 37). Its 
essential parts are: a Heidenhain thermo- 
meter ..(D) graduated in hundredths and 
reading from — 1° to — 5° C; a platinum 
wire loop for stirring (E); a test-tube (A) 
which is closed by a stopper through which 
the thermometer and stirring wire pass, and 
which in turn is placed in a second larger 
tube (B) so as to be surrounded by an air 
space. The jar (C) is filled with a freezing 
mixture of salt and ice, the temperature of 
which should lie between — 2° and — 5° C. 
Into this is placed the second tube B. The 
test-tube A is charged with 20 c.c. of blood 
(if only 10 c.c. are available, this amount 
may suffice), obtained by means of a large 
aspirating syringe from one of the veins near 
the bend of the elbow, as in the case of a 
bacteriological examination of the blood ; the 
thermometer is introduced and the stirring 
wire adjusted. The tube is placed directly 
in the freezing mixture until the mercury 
leaves the reservoir bulb (F) ; this is done 
to save time. It is then adjusted in the second 
tube, as shown in the illustration, and the 
blood constantly stirred with the platinum 
wire. The temperature falls more or less 
rapidly below the freezing point before 
actual freezing takes place; as this occurs it suddenly rises again 
owing to liberation of heat, and then remains constant for some 
time. This point represents the true freezing point. Later, if the 
tube is allowed to remain in the freezing mixture, the temperature 
may fall to that of the latter. The difference between the freezing 
point of distilled water and that of the blood is d. 




Fig. 37. — Beckmann's 
apparatus. 



STUDY OF OSMOTIC RESISTANCE OF THE BED CELLS 157 

In every case it is necessary to determine the true zero for each 
instrument separately, as this often varies somewhat owing to un- 
avoidable errors incident to its construction. To this end the tube 
A is charged with three to four times the amount of distilled water 
which is necessary for one examination. The greater portion of this 
is frozen; the liquid portion is thrown away; the frozen water is 
allowed to thaw and is again frozen in part, a portion being again 
thrown away; the remainder is sufficiently pure for the examination. 

The freezing mixture is prepared by packing alternate layers of 
ice and salt into the jar around the tube B, which is held in position 
while the ice is packed. Ice and salt are finally thoroughly mixed 
by stirring with a heavy wire ring and rod (G). If several exami- 
nations are to be made, the water which separates out is poured off 
and replaced by an additional amount of salt and ice. 

The method is quite expeditious, and if everything is previously 
prepared the examination does not occupy more than ten or fifteen 
minutes. 

Literature. — v. Koranyi, Zeit. f. klin. Med., 1897, vol. xxxiii, and 1898, vol. 
xxxiv. Lindemann, Deutsch. Arch. f. klin. Med., 1899, vol. lxv. Albarron, 
Annal. d. mal. genito-urin., 1899. Senator, Deutsch. med. Woch., 1900, vol. xxvi, 
p. 48. Claude and Balthazar, Presse med., 1900, vol. xviii, p. 85. Casper and 
Richter, Funktionelle Nierendiagnostik, Berlin, u. Wien, 1901. Kiimmel, Cen- 
tralbl. f. Chir., 1902, vol. xxix, p. 121 of Beilage. Tinker, Johns Hopkins Hosp. 
Bull., 1903, vol. xiv, p. 162. 



STUDY OF THE OSMOTIC RESISTANCE OF THE RED CELLS. 

Janowsky's Method. — The red cells are first counted as usual, 
using a 3 per cent, sodium chloride solution as diluent. Then a second 
count is made; this time with a hypotonic (0.4 per cent.) salt solution 
and a dilution of 1 to 200. Ten minutes should be allowed to elapse 
before mounting the drop. At the end of this time even normally a 
certain number of red cells lose their hemoglobin. This number is 
expressed in percentage terms, pro 1 cb. mm. of blood. The exami- 
nation should always be made upon an empty stomach and in accurate 
work the barometric pressure and in cases of heart disease the height 
of the blood pressure should also be taken into consideration. 

Under normal conditions the corpuscular stability is subject to 
definite individual variations, which lie within very narrow limits. 
It is increased by physical and mental labor, diminished by baths and 
diet free from meats. 

Jakuschewsky 1 found normal values in diabetes (excepting in 
coma), pseudoleukemia, the primary stages of syphilis, chronic gas- 
tritis, atrophic hepatic cirrhosis, subacute parenchymatous nephritis, 
pyelonephritis, hysteria, and minor chorea. In aortic aneurism the 

1 Russ. med. Rundsch, 1904, p. 345 



158 THE BLOOD 

stability is high, but quite analogous to what is found in normal old 
people with physiological sclerosis. 

Increased stability associated with an increase in the severity of 
the clinical symptoms and vice versa was noted in the following con- 
ditions: typhoid and typhus fever, recurrens, croupous pneumonia, 
acute and chronic malaria, influenza, acute rheumatism, advanced 
pulmonary tuberculosis, intestinal tuberculosis; chronic parenchy- 
matous and interstitial nephritis (in association with uremic symp- 
toms); anemia, chlorosis, leukemia, catarrhal jaundice; Charcot- 
Hanot's (biliary) cirrhosis; attacks of cholelithiasis with bile retention; 
acute gout; myocarditis (with beginning insufficiency); organic heart 
disease during lack of compensation; the final stage of carcinoma of 
the stomach. 

Jakuschewsky thinks that the determination of the corpuscular 
stability may be of prognostic significance — an increase or retarded 
diminution ceteris "paribus indicating an aggravation of the condition — 
and at times also of diagnostic value (carcinoma of the stomach). 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 

General Technique. — In order to obtain results of value it is 
usually necessary to procure the blood for bacteriological examination 
directly from a bloodvessel. To this end the most prominent super- 




Fig. 38. — Blood aspirator; half size. (Ewing.) 

ficial vein near the bend of the forearm is chosen. An hour or two 
before puncture the entire region of the arm is thoroughly scrubbed 
with tincture of green soap, rinsed with warm sterile water, and finally 
washed with alcohol and with ether. A bichloride compress (1 to 500) 
is applied and left in situ until everything is ready for aspiration. It 
is then removed, and the area thoroughly rinsed and scrubbed with 
sterile water. An assistant compresses the large bloodvessels above 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 159 

the elbow with sufficient force to bring the superficial veins out 
prominently, but not to arrest the flow of blood. (A band firmly 
applied answers the same purpose.) For aspirating purposes the 
instrument pictured in the accompanying figure is more convenient 
than a hypodermic syringe. The tube is of about 10 c.c. capacity 
and graduated in c.c; it is ground at one end so as to fit a No. 42 
hypodermic needle. The glass tube contains a small plug of cotton 
at the far end. Needle and tube (minus rubber tube) are sterilized 
in a large test-tube by dry heat. When cool the rubber tube is slipped 
on. The needle is thrust obliquely into the most superficial vein 
(median basilic), being held almost parallel to the vessel. This is 
facilitated by steadying the bloodvessel with the fingers of the other 
hand (asepsis!). Blood flows immediately and this can be hastened 
by gentle aspiration. After the operation a small pledget of bichlo- 
ride gauze is placed upon the site of the puncutre. As a rule the 
patients complain but little of pain, but in nervous persons a little 
ethyl chloride spray may be advantageously employed. 

The contents of the tube are then at once divided among the culture 
media as described in detail below. 



rM 


El> *t 


Mb 


M * 


/ £gt 


mSb 




c ■ 


'9)1 


m^ 




» 

■4 


!'>v ** 


*tj * 




' m ■ & 



Fig. 39.— Positive agglutination reaction. 

Typhoid Fever. — Recent research has shown that in typhoid fever 
the specific organism (Fig. 39) can be isolated from the blood in 
a large percentage of cases (over 80 per cent.) and at a time when 
the Widal reaction (see below) may not yet be demonstrable. In 
cases of great as well as moderate severity the organism is usually 
demonstrable during the entire febrile period, as also ^during 
relapses. It has hence been suggested that a bacteriological blood 



J 60 THE BLOOD 

examination be made in all doubtful cases. The method is thor- 
oughly practicable and deserves general recognition. 

Method. — 8 to 10 c.c. of blood are withdrawn from one of the 
superficial veins of the arm as described. Several Erlenmeyer flasks, 
each containing 150 c.c. of bouillon, should be ready at hand. Blood 
is added to these in varying proportions : two receive 1 c.c. each and 
two others 2 c.c. each. In this way 1 to 150 and 1 to 75 dilutions are 
obtained. The flasks are well shaken and placed in the incubator 
for twenty-four hours. A hanging drop is then examined. 1 If 
negative the incubation is continued for twenty-four hours further. 
When the bouillon has become cloudy, subcultures are made in 
milk and glucose bouillon (see description of typhoid bacillus) and 
the organism further tested with an actively agglutinating serum (see 
below). 

It is interesting to note, however, that the tendency to agglutina- 
tion of freshly isolated typhoid bacilli is almost invariably much 
inferior to that of bacilli which have been maintained for a long 
time on artificial media. Courmont thus notes that they were 
commonly agglutinated with a dilution of 1 to 50 by a serum which 
agglutinated laboratory bacilli at 1 to 200. 

Literature. — Neuhaus, Berlin, klin. Woch., 1886, Nos. 6 and 24. Schott- 
miiller, Deutsch. med. Woch., 1900, No. 32. Castellani, cited in Presse med., 
June, 1900. Auerbach u. Unger, Deutsch. med. Woch., 1900, No. 29. Cole, 
Johns Hopkins Hosp. Bull., 1901, p. 203. Courmont. Jour. d. physiol. et d. 
pathol. gen., 1902, vol. iv, p. 155. Polacco and Gemelli, Centralbl. f. inn. Med., 
1902, vol. xxiii, p. 121. 

Agglutination Test (Pfeiffer-Widal Reaction). — Owing to the develop- 
ment of specific agglutinins in the blood serum of typhoid patients, as a 
consequence of infection, such serum possesses the property of causing 
arrest of motility and agglutination of the corresponding bacilli. This 
observation, originally made by Pfeiffer, was utilized for diagnostic 
purposes by Widal, in 1896. The method which bears his name has 
been generally adopted in the clinical laboratory, and must be regarded 
as a most valuable aid in the diagnosis of typhoid fever. The reaction 
occurs in over 95 per cent, of undoubted cases, and may appear as 
early as the first day of the disease, meaning thereby the first day 
that the patient spends in bed or the fifth day of general malaise. 
Such instances, however, are uncommon, and, as a general rule, 
a positive result is obtained only after the fifth or sixth day in bed. 
In a small number of positive cases, on the other hand, the patient 
may pass through the entire course of the disease, and present typ- 
ical clumping only during convalescence or a subsequent relapse. In 
every case, therefore, in which no reaction is obtained upon first trial, 
the test should be repeated at regular intervals throughout the disease. 

1 At first the bacilli are but little active, but on further cultivation and rein- 
oculation their motility increases 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 161 

Intermittence of the reaction, moreover, is very common, and empha- 
sizes still further the necessity of frequent examinations in apparently 
negative cases. 

While in some instances the reaction disappears very soon after 
the temperature reaches normal, and even earlier, it generally con- 
tinues into convalescence, and may in some cases be observed months 
and years after the attack. Cases have been recorded in which a 
positive reaction could be obtained thirty-seven years after infection. 

In a series of 71 cases who had had typhoid fever in the past Krause 1 
found the reaction in 36 — in 1 instance twelve years after the illness. 
Of 26 cases examined within a year 16 gave a positive result, of 21 
from the second to the fifth year 12, of 19 from the fifth to the tenth 
year 7, and of 5 from the tenth to the twentieth year 1. In 3 in- 
stances no reaction could be obtained within a month of the disease. 

Such observations, of course, entail the usefulness of the test to a 
certain extent. For this reason the demonstration of a negative 
reaction early in a case which is followed by a positive reaction is a 
particularly valuable symptom, the early negative result eliminating 
the possibility that the subsequent positive finding could be referable 
to an antecedent typhoid. 

The question whether or not Widal's reaction is a specific reac- 
tion of the typhoid organism can be answered in the affirmative, 
notwithstanding the fact that at times cases of apparently true typhoid 
fever are seen in which no clumping is obtained, and that the reaction 
has been observed in cases which were apparently non-typhoid. 
Such exceptions are due in part to faulty technique, viz., to too low a 
degree of dilution of the serum, the use of old or impure cultures, too 
long a time limit of observation, single negative tests, etc. On the 
other hand, there can be no doubt that typhoid bacilli are at times 
present in the body without giving rise to symptoms of typhoid fever. 
In a case of cholelithiasis, reported by Cushing, typhoid bacilli were 
found in the gall-bladder, and distinct clumping was observed with 
a dilution of 1 to 30, although no history of typhoid fever could be 
obtained. Another interesting apparent exception to the rule that the 
Widal reaction is only obtained in cases of typhoid infection is re- 
ported by Grtinbaum, 2 who notes that he obtained a positive reaction 
in cases of febrile jaundice. His observations have since been amply 
confirmed. The biliary components pro se have manifestly nothing 
to do with the production of the reaction, however, as is shown by 
the observation of Kammerer 3 who obtained agglutination in only 3 
jaundice cases out of 50 (from the most diverse hepatic diseases). In 
the positive cases no doubt infection had occurred by some organism 

1 Zentralbl. f. Bact., 1904, vol. xxxvi, No. 1. 

2 Cited bv Durham, Brit, Med. Jour., 1898, vol. ii, p. 600. 

3 Berlin, klin. Woch.. No. 2.5, 1904. 

It 



162 THE BLOOD 

of the paratyphoid group, standing nearer to the typhoid than the 
colon bacillus. 

There can further be no doubt that individuals exist who are 
naturally immune against typhoid fever, and that some of the positive 
results which have been obtained in healthy individuals who have 
never had typhoid fever may be explained in this manner. 

So far as the non-occurrence of the reaction in cases of apparently 
true typhoid fever is concerned we now know that infections occur 
with organisms which are closely related to the typhoid bacillus 
(the paratyphoid group) and which clinically resemble typhoid 
fever, but which give no reaction with the typhoid bacillus, unless in 
low dilutions. (See Paratyphoid Fever.) 

WidaVs test is a most valuable aid in the diagnosis of typhoid fever, 
but cannot be relied upon to the exclusion of other symptoms. 

Technique. — The method is based upon the fact that typhoid 
serum will cause arrest of motility and agglutination of the specific 
bacilli even when diluted, whereas clumping of the same organism is 
obtained only with sera from other diseases and healthy individuals 
when these are used in a more concentrated form. The time limit 
at which clumping occurs is likewise an important factor, as non- 
typhoid sera are at times met with in which, notwithstanding a 
certain degree of dilution, agglutination occurs, providing that the 
specimen is kept for a long time. Both factors — viz., the degree of 
dilution necessary to eliminate the agglutinating power of non-typhoid 
sera, as also the time limit of observation — have been arbitrarily de- 
termined. Widal originally advised a dilution of 1 to 10, and Gruber 
a time limit of one-half hour. It was soon ascertained, however, 
that this dilution was too low, and most observers have favored a 
dilution of 1 to 40 or 1 to 50. At the present time there is a ten- 
dency to further increase this even as far as 1 to 200 with a time 
limit of one-half hour. 

With the original method only a full virulent, fresh bouillon culture 
of the typhoid bacillus, viz., one not older than sixteen to twenty- 
four hours, is employed. The further technique is simple : 1 volume 
of blood serum is diluted with the requisite amount of normal salt 
solution to 20, 25, 50, or 100 volumes, as the case may be. Of this 
mixture one droplet is mounted on a cover-glass, mixed with a droplet 
of the typhoid culture (dilutions of 40, 50, 100, or 200 thus resulting), 
and inverted over a cupped slide, with a little vaselin along the edges. 
The examination is conducted with a medium power (Leitz, 6 or 7; 
Bausch and Lomb, -J-). 

If the case in question is one of typhoid fever, it will be observed 
that after a variable length of time the individual bacilli, which at 
first actively dart about the field of vision, become quiescent and tend 
to gather in distinct clumps, while the interspaces become entirely 
free from bacilli or very nearly so. After one-half hour, or one 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 163 



or two hours, according to the degree of dilution, all motion has 
ceased. When the time limit has expired and loss of motility and 
agglutination have not occurred the result is negative. In such an 
event further examinations should be made on the following days. 
In every case it is well to make a control test with the simple bouillon 
culture, so as to ensure the absence of preformed clumps and the 
virulence of the organism ; of the latter, the degree of motility is the 
best index. In order to secure the 
necessary degree of dilution, various 
methods have been suggested. The 
simplest, and the one generally em- 
ployed in municipal bacteriological 
laboratories, is to receive a large drop 
of blood upon a slide or slip of glazed 
paper, and allow it to dry. A drop or 
two of distilled water is then placed on 
the blood and allowed to remain for sev- 
eral minutes, when it is further diluted 
and examined as described. The 
principal advantage of this method is 
its simplicity and the fact that the dried 
blood retains its agglutinating proper- 
ties for weeks and months. The 25Cmm 
results, however, are less reliable than 
with the use of liquid blood. This 
can be readily collected in little glass 
capsules, such as Wright has recom- 
mended for opsonic work (Fig. 40, b). 
The finger or ear is pricked as usual 
and the blood allowed to enter the 
bent capillary arm of the capsule by 
merely being held in contact. When 
enough has been collected, the far end 
of the capsule is warmed and the 
straight end sealed, when the blood will 
mount into the body of the capsule. 
The bent arm is then also sealed. In 
this manner the blood can be kept for 
a long time. At the laboratory it is 
hung into the centrifuge, if the serum 
has not already separated out, briefly centrifugated, and the capsule 
cut with a file. The serum is then diluted with the aid of a Thoma- 
Zeiss pipette or a common capillary pipette such as anyone can 
construct and is pictured in Fig. 40. These pipettes are destroyed 
after use. 

A very material advance in the practical application of the agglutina- 



20 



/5 



/0 



5 




Fig. 40. — a, Wright's pipette; b, Wright' 
blood capsule. 



164 THE BLOOD 

tion test was made by the discovery that it is not necessary to work 
with living cultures of the typhoid bacillus, but that dead bacilli 
will answer just as well, providing they are killed off when in 
a virulent condition. To this end formalized cultures are espe- 
cially convenient. To prepare this a twenty-four to forty-eight hours' 
bouillon culture of an actively agglutinable strain is treated with 
formalin to the extent of 1 per cent, of the solution, and set aside for a 
week. The bacteria are allowed to settle, when the supernatant 
fluid is poured off and replaced by formalized normal salt solution. 
In this form the material will keep for months. Before use it should 
be well agitated and examined to see that no artificial clumps are 
present. With the formalized culture the microscopic examination 
can then be made, or one can proceed macroscopically. If the micro- 
scopic test is used the examination is made after two to twenty 
hours. 

The so-called Ficker Diagnosticum is a suspension of typhoid 
bacilli which have been killed off by a special process, which has 
not been made public. The outfit is sold by Merck and is used in 
the macroscopic application of the test. It consists of a series of 
small corked tubes, a graduated dropping tube, a bottle of the diag- 
nosticum and one of normal salt solution, a small cupping glass and 
lancet. Cupping glass, rubber stopper, and lancet must first be steril- 
ized by boiling in water. The blood is obtained from the back of the 
patient by making three or four deep punctures 1 and applying the 
cupping glass in the usual manner, viz., after placing a few drops of 
alcohol in the bottom and igniting it and rapidly placing the bottle 
to the skin before the flame is extinguished. The skin of the back 
is first cleansed with soap and water, alcohol, and ether. About J. c.c. 
of blood is thus drawn, the bottle closed with the rubber cork and set 
aside in a cool place until the serum has separated. The test-tube 
and pipette are sterilized by means of alcohol and ether and the corks by 
boiling in water; 0.1 c.c. of the clear serum is now placed in one of the 
test-tubes, and after washing the pipette with water, alcohol, and ether, 
diluted with 0.9 c.c. of normal salt solution. A dilution of 1 to 10 thus 
results. The mixture is well shaken, and 0.1 c.c. placed in a second 
tube and 0.2 c.c. in a third. With the carefully washed pipette 0.9 
c.c. of the diagnosticum is added to test-tube No. 2 and 0.8 c.c. to No. 3. 
Dilutions of 1 to 100 and lto 50 thus result. A further tube (No. 4) 
receives 1 c.c. of the diagnosticum alone. All tubes are closed, well 
agitated, and set aside in the dark at room temperature. They are 
inspected after ten to twelve hours, when as a rule a positive reaction 
can be detected. Sometimes it is necessary to wait for twenty 
hours; if after that the result is negative it is so reported. If the 
reaction is positive the bacilli in tubes 2 and 3 will have fallen to the 

1 I find it more convenient to collect the necessary amount of blood from the 
ear; from X to 5 c.c, can be obtained by ordinary puncture without difficulty. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 165 

bottom, leaving the supernatant fluid clear, while the control tube 4 
remains turbid. All tubes should be viewed against a dark back- 
ground. 

The results which are obtained with Ficker's diagnosticum are very 
satisfactory. The method has been amply investigated and uniformly 
endorsed. 

The formalized cultures described above can be utilized just as 
well as the diagnosticum and in the same manner or any other modifi- 
cation that may suggest itself to the individual worker 1 . Capillary 
pipettes such as the one pictured in Fig. 40, a, can be used in the 
place of the special, calibrated instrument mentioned. 

Paratyphoid Fever. — In cases of so-called paratyphoid fever 
organisms may be met with in the blood which apparently occupy a 
position intermediate between the typhoid bacillus and the organisms 
belonging to the colon group. Collectively they are spoken of as 
paratyphoid bacilli, though the question whether or not they represent 
well-defined species has not been definitely settled. 

Cases of paratyphoid fever clinically resemble true typhoid, but 
are on the whole milder in their course. As a rule the serum does 
not react with the typhoid bacillus, while the organism which appears 
to be pathogenic in the individual case is agglutinated in a typical 
manner. Unfortunately, however, the serum of one case will not 
always react with the organism of a second case; so that the serum 
reaction will not always make it possible to distinguish the inter- 
mediates as a group from typhoid on the one hand, and the bacillus 
coli on the other. 2 Moreover, it has been shown that the serum of 
true typhoid may agglutinate the paratyphoid bacillus in higher 
dilutions even than the typhoid bacillus, although this is probably 
not usual (Griinberg and Roily). 

The paratyphoid group includes WidaPs paracolon bacillus, 
Gwyn's bacillus, Cushing's bacillus 0, Hewlett's bacillus b, Noonan's 
bacillus, Schottmiiller's bacilli, etc. It is subdivided into two groups, 
A and B, of which B causes at first an acid reaction in litmus milk, 
which in about ten days changes to a permanently alkaline reaction, 
while group A causes permanent acidity. Unlike . the typhoid 
bacillus the paratyphoid ferments dextrose with the formation of 
gas. The intermediates do not form gas in lactose nor in saccharose 
media. On potato the growth is slight and there is no discoloration. 
They do not ferment milk or produce indol. 

The examination of the blood is conducted as in typhoid fever, 
but it is not always necessary to dilute it to the same degree. Some- 
times successful cultivation follows the spreading of a few c.c. of 
blood over the surface of agar tubes or plates. 

1 Riidiger, Jour. Infect. Dis., 1904, vol. i, p. 236. 

2 This only holds good for members of the paracolon group, while those of 
the paratyphoid groups interact without exception. 



166 THE BLOOD 

Literature. — Gwyn, Johns Hopkins Hosp. Bull., 1898, vol. ix. p. 54. Cushing, 
ibid., 1900, vol. xi, p. 156. Schottmuller, Zeit. f. Hyg., 1901, vol. xxxviii. W. B. 
Johnston, Am. Jour. Med. Sci., 1902, vol. cxxiii, p. 187 (analysis of all cases reported 
up to that time). A. W. Hewlett, ibid., p. 200. Coleman-Buxton, ibid., 1903, vol. 
cxxiii, p. 979. See also Ascoli, Zeit. f. klin. Med., 1903, vol. xlviii, p. 419. 

Pneumonia. — According to Rosenow the pneumococcus can be 
recovered in practically all cases of pneumonia, providing that large 
quantities of blood are used. He does not think that their presence 
indicates an especially unfavorable prognosis. He obtained positive 
results in 160 of 175 cases with a mortality of 40 per cent. 

Positive results were obtained as early as twelve hours after the 
initial chill and as late as forty-eight hours after the crisis, although 
negative results are the rule after crisis. Pneumococci were also 
obtained in the blood smears in 47 out of 80 cases. The results of 
other modern investigators are similar. 

Prochaska, working under Eichhorst's direction, found pneu- 
mococci in the blood in each of 10 cases examined, and in a sub- 
sequent series of 40 cases, of which 7 were fatal, he obtained the 
pneumococcus in 38. 

Frankel states that according to his experience, which is based 
upon an examination of more than 150 cases, one may infer that 
death will occur either with the symptoms of sepsis or that metas- 
tasis will take place in the internal organs whenever a larger number 
of colonies develop on spreading 1 c.c. of blood upon a plate of 
agar. If, however, the number is so small that it is necessary to 
take larger amounts of blood to demonstrate their presence and to 
grow them in bouillon instead of on agar, so as to eliminate the bac- 
tericidal power of the blood altogether, then Frankel believes their 
presence is of no significance, and does not warrant a fatal progno- 
sis. In the latter case he has found that the bacteria are frequently 
avirulent. 

The examination, which should be repeated every day, if necessary, 
is conducted as follows: After disinfection of the arm in the usual 
manner 10 c.c. of blood are aspirated and agar tubes — liquefied at 
40° C. — inoculated, each with 1 or more c.c. of the blood. Plates are 
then prepared and kept at a temperature of from 35° to 37° C. The 
colonies appear as small, round, grayish, jelly-like drops, which are 
quite characteristic. 

Rosenow finds that the best results are obtained with blood agar. 
Upon this the pneumococci, especially when very virulent, produce 
a hemolytic zone which is greenish in color. This phenomenon, 
according to Schottmuuller, may serve to distinguish the pneumococcus 
from streptococci, which cause hemolysis without pigment production. 

Instead of agar, bouillon may also be employed, and it is quite 
likely, as Prochaska suggests, that in this manner positive results 
may be more frequently obtained. Cole recommends the use of 
sterile litmus milk, of which portions of 150 c.c. each are employed 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 167 

in Erlenmeyer flasks. Early acidification and coagulation occur, 
and it is thus possible to determine more readily and quickly whether 
growth has taken place. Smears are then made and examined for 
capsules (see below). The identity is established by the characteristic 
shape and staining reactions of the organism, including the staining 
of the capsules, by the typical growth in milk and agar, and by the 
absence of growth, or very slight growth, in gelatin at ordinary room 
temperature. Especially characteristic, further, is the fermentation 
of inulin by the pneumococcus. To this end serum water containing 
inulin is used as recommended by Hiss. 

The individual organism (Fig. 41) is capsulated, and usually 
occurs in pairs, arranged end to end or in short chains. At 
times, however, the chains are quite long, and then it may be difficult 




Fig. 41.— Pneumococcus, showing capsule. 



to distinguish it from streptococci. It is easily stained with the com- 
mon aniline dyes. In order to differentiate the capsule, the method 
suggested by Epstein (see Sputum) should be employed. It should 
be remembered that capsules are only demonstrable in specimens 
obtained from milk or blood-serum cultures, while they are not 
shown in growths obtained from agar or bouillon. 

Agglutination. — According to Rosenow pneumococcic serum in- 
variably agglutinates the pneumococcus with a maximum dilution of 
1 to 40 or 1 to 50. With rabbit-immune serum and using his serum- 
water medium for the growth of the organism, Hiss obtained aggluti- 
nation with dilutions up to 800 and over. 

Literature. — Goldscheider, Deutsch. med. Woch., 1892, No. 14. Sittmann, 
Deutsch. Arch. f. klin. Med., 1894, vol. liii, p. 323. Kiihnau, Zeit. f. Hyg., 1897, 
vol. xxv. Kohn, Deutsch. med. Woch., 1897, p. 136. James and Tuttle, N. Y. 
Presbyterian Hosp. Rep., vol. iii, p. 44. Sello, Zeit. f. klin. Med., 1898, vol. 
xxxvi. White, Jour, of Exper. Med., 1899, vol. ii. Silvestrini and* Sertoli, 
Riforma Med., 1899, No. 116. Abstr. in Centralbl. f. inn. Med., 1899, vol. xxi. 



168 THE BLOOD 

R. Cole, Johns Hopkins Hosp. Bull., 1902, vol. xiii, p. 236. Prochaska, Centralbl. 
f. gen. Med. 1900, No. 46. Prochaska, Deutsch. Arch. f. klin. Med., vol. lxx, p. 
559. Frankel, Deutsch. med. Woch., 1901, V. B., p. 212. Rosenow, Jour. 
Amer. Med. Assoc, 1905, No. 2, p. 851; Jour. Infect. Dis., March, 1904. 

Pyogenic Bacteriemia. Technique. — The general technique is the 
same as that described before, but large amounts of blood are advised, 
viz., 20 to 25 c.c. The media which are commonly employed are the 
ordinary laboratory media; in addition Libman has suggested the 
use of serum-glucose agar and serum-glucose bouillon. He has 
pointed out that on these media the growth of most bacteria is more 
marked and more rapid than on ordinary serum agar. This is true 
especially of the streptococcus, the pneumococcus, the gonococcus, 
and the meningococcus. With the solid media plates are employed 
almost altogether to the exclusion of media in tubes; 2 to 3 c.c. of 
blood are used for 15 to 20 c.c. of the solid media. 

The number of organisms which may be found in the blood in 
septic conditions is exceedingly variable. On the one hand, but one 
plate or flask out of several may show any growth, and then only 
after several days; while, on the other hand, the number of organ- 
isms may be quite large. Cole has reported a case of streptococcus 
septicemia in which the number of organisms amounted to 3642 
per cubic centimeter of blood six days before death, and then rose 
to 10,716 per cubic centimeter two days before death. I have seen 
a case of meningococcus septicemia in which the organisms numbered 
7,380,000 per cubic centimeter just before death. 

The time before death at which organisms may be found in the 
blood is also quite variable; sometimes they may be demonstrable 
a month before, in other cases only a day or two before the fatal 
issue. 

(a) Staphylococcus Bacteriemia. — Staphylococcus bacteriemia is more 
common than was formerly supposed. The variety usually seen is 
the Staphylococcus aureus. The albus is rare. Libman states that 
the latter plays an insignificant role in systemic infections; that in 
several years he has not met with a single instance in which he could 
ascribe a systemic infection to the Staphylococcus albus. He draws 
attention to the fact that the pigment production in the aureus may 
be delayed and that some of the positive albus cases recorded in the 
literature may in reality have been aureus cases of this kind. He 
accordingly recommends that an apparent albus be observed for five 
days and grown upon potato and serum agar before the diagnosis is 
made (glucose interferes with pigment production). Staphylococcus 
citreus also is very rare. 

Of the 28 positive findings in Libman's large series of blood cultures 
many were instances of osteomyelitis, some were secondary to furun- 
cles or cellulitis, others were cryptogenetic, and 2 referable to post- 
partum* infection (rare). All these were aureus cases. The only 
positive albus cases were obtained within forty-eight hours before 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 169 

death; Libman looks upon these as agonal invasions. The Staphy- 
lococcus citreus was isolated once in a case of osteomyelitis. 

F. Meyer and Michaelis and others report having found pus 
organisms in a large percentage of cases of advanced phthisis. This 
is denied by Jochmann, excepting as agonal infections. 

The Staphylococcus pyogenes aureus occurs in the form of spheri- 
cal bodies, averaging about 0.8 fx in diameter, which readily stain 
with the basic aniline dyes, as also with Gram's method. They 
usually occur in clumps, but may also be seen in pairs and in short 
chains. The organism grows on all culture media, and in the pres- 
ence of oxygen gives rise to the formation of an orange-yellow pig- 
ment. Gelatin is rapidly liquefied; it coagulates milk with acid 
reaction and clouds bouillon. The Staphylococcus pyogenes albus 
and citreus differ from the aureus by the absence of pigment in the 
first and by the formation of a lemon-yellow pigment in the second. 





V / 




Fig. 42.— Streptococcus pyogenes. X 800. (Frankel.) 

(b) Streptococcus Bacteriemia. — In the large series of blood cultures 
reported by Libman streptococci were isolated in 58 cases. Strepto- 
coccus bacteriemia is thus more common than staphylococcus bacteri- 
emia. Some were instances of terminal infections, or infections aris- 
ing from the tonsils, the ears, and mastoid processes, or the genito- 
urinary tract (abortion and postpartum infections), while in others 
infections were referable to wounds, and still others were cryptogenetic. 
Some cases were characterized by joint or bone lesions. Endocar- 
ditis was frequent. One was a case of erythema nodosum. In several 
cases of mild acute endocarditis, following what clinically appeared 
to be typical articular rheumatism, Libman found attenuated strepto- 
cocci. They could be demonstrated during extended periods of time. 

Streptococci have also been found in the blood in advanced cases of 
phthisis (probably as agonal invasions). 

Hektoen has pointed out that in scarlatina streptococci may be 
found in the blood during life in at least 18 per cent, of all cases. 



170 THE BLOOD 

I append his conclusions: Streptococci may occasionally be found 
in the blood of scarlet-fever cases that run a short, mild, and uncompli- 
cated clinical course. They occur with relatively greater frequency 
in the more severe and protracted cases of the disease, in which there 
may also develop local complications and clinical signs of general 
infection, such as joint inflammations; but even in the grave cases 
of this kind spontaneous recovery may take place. In fatal cases 
streptococci may not be demonstrable. The theory that scarlet fever 
is a streptococcus disease thus does not seem to receive direct support 
from these investigations. 

In diphtheria, measles, and smallpox infection with streptococci is 
also not uncommon. Other organisms may, however, also be met 
with, such as the various staphylococci, and quite commonly also, 
according to Jehle, the bacillus of influenza. 

The Streptococcus pyogenes (Fig. 42) occurs in chains of spherical 
cocci which usually vary from four to twenty in number. The size 
of the individual organism is somewhat greater than that of the 
staphylococcus, but may vary even in one and the same chain. It 
is readily stained with the basic aniline dyes and also with Gram's 
method. It grows on all culture media at the temperature of the 
room, forming small, gray, granular colonies on agar and gelatin. 
Unlike the pneumococcus it does not ferment inulin media. As 
a rule, it does not liquefy gelatin, and it may or may not coagulate 
milk and cloud bouillon. Several varieties are recognized, viz., 
Streptococcus brevis, which forms short chains; Streptococcus longus, 
which occurs in long chains; streptococci, which render bouillon 
cloudy, and those which do not; streptococci, which form flocculent, 
sandy, scaly, or viscous sediments. 

The Streptococcus conglomeratus grows, without clouding bouillon, 
in the form of dense, separate particles, scales, or thin mebmranes 
at the bottom and sides of the tube, and on shaking the sediment 
it breaks up into little specks, without producing uniform, diffuse 
cloudiness. The chains are long and interwoven in conglomerate 
masses (Welch). 

(c) Non-pneumonic Pneumococcus Bacteriemia. — In Libman's series, 
apart from the pneumonia cases, pneumococci were found only 
four times. Twice there was an acute endocarditis of unknown 
source, once there was an infection between two toes, and once there 
was a suppurating ethmoiditis and frontal sinusitis, with abscess. 
Other observers have found the organism in cases of biliary abscess 
at the time of the chill, in suppurative oophoritis, in peritonitis, 
etc. It is interesting to note in this connection that Libman obtained 
only negative results in 25 cases of peritonitis, and also in a series of 
25 cases of appendicitis, some of which were very severe. 

(d) Bacterium Proteus Bacteriemia. — Libman reports a case of 
uremia in which the proteus was found one day before death, 
together with streptococci. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 171 

(e) Colon Bacillus Bacteriemia. — The colon bacillus also is rarely 
found in the blood. Libman mentions a case in which it was 
demonstrated where the operation of internal urethrotomy had 
been performed. An interesting case is also reported by Rochester 
(see literature below). 

(f) Paracolon Bacteriemia. — Aside from those cases in which para- 
colon bacilli have been found in so-called paratyphoid fever, para- 
colon bacteriemia is very rare. Libman and Berg report one case 
which clinically resembled cholecystitis. 

(g) Bacillus Pyocyaneus Bacteriemia. — The Bacillus pyocyaneus 
is rarely found in the blood. Libman and Brill report a case in which 
it occurred secondarily to a Staphylococcus aureus bacteriemia. 

(h) Gonococcus Bacteriemia. — Cases of gonorrheal septicemia in 
which the gonococcus was isolated from the blood of the patients 
during life have been reported by Thayer-Blumer, Thayer-Lazear, 
Byelogoway, Wilson, Harris-Johnston, and others. In all these cases 
gonorrheal endocarditis existed. In other infections of the same 
nature positive results were obtained by Ahmann, Colombini, Panichi, 
and Unger, in association with polyarthritis, epididymitis, myositis, 
tendovaginitis, inguinal bubo, and parotitis. In the endocarditis 
cases cultures were obtained after an illness lasting for from five 
weeks to seven months, at times as early as the ninth to the eleventh 
day preceding death, and on an average five days before death. 

To cultivate the gonococcus from the blood during life, it is neither 
necessary to use a large amount of blood nor to dilute it greatly, 
nor to employ any specially prepared medium. From 2 to 5 c.c. are 
sufficient. According to Harris and Johnston, it is more advan- 
tageous to mix the blood with melted agar and to plate the same 
than to use fluid media where the oxygen supply is more restricted. 

(For a description of the organism, see Gonorrheal Pus.) 

Literature. — N. M. Harris and W. B. Johnston, " Gonorrhoeal Endocarditis 
with Cultivation of the Specific Organism from the Blood during Life," Johns 
Hopkins Hosp. Bull., 1902, vol. xiii, p. 236 (literature). Thayer and Blumer, 
ibid., 1896, vol. vi, p. 59. Thayer and Lazear, Jour. Exper. Med., vol. iv, p. 81. 

(i) Micrococcus Zymogenes Bacteriemia. — This organism is appar- 
ently closely related to the Pneumococcus and the Streptococcus 
pyogenes. It has been isolated from the blood in one instance 
by MacCallum and Hastings. 

(k) Meningococcus Bacteriemia. — In several instances of meningo- 
coccus meningitis the corresponding organism has been isolated from 
the blood. In one case I found 7,380,000 diplococci per cubic 
centimeter. The organisms could be demonstrated in large num- 
bers directly in the blood smear. Almost all were enclosed in polynu- 
clear neutrophiles and in large mononuclear elements. 

Endocarditis. — In acute endocarditis or in acute exacerbations 
of chronic cases bacteriemia is fairly common. Lenhartz obtained 



172 THE BLOOD 

positive results intra vitam in 16 cases out of 28, and Libman states 
that in cases of acute ulcerative endocarditis he has always found 
organisms in the blood. The organisms which have been encountered 
are the Staphylococcus aureus, streptococci, pneumococci and the 
gonococcus. Of these the streptococcus cases are the most common, 
while the staphylococcus comes next in order. Pneumococcus and 
gonococcus endocarditis is relatively uncommon. Libman remarks 
that there is often a marked disproportion between the number of 
bacteria in the blood and the extent of the lesion. There may be an 
almost countless number in the blood and only very small deposits on 
the valves, or there may be large vegetations with hardly any bacteria 
in the blood. As a rule they are present in fair numbers. 

In a series of 10 cases of mild acute endocarditis following what 
clinically appeared to be typical articular rheumatism Libman 
could demonstrate attenuated streptococci and diplococci during 
extended periods of time. 

Prognosis in Pyogenic Bacteriemia.— The prognosis in the 
pyogenic bacteriemias, aside from other considerations, is upon 
the whole unfavorable; recoveries, however, are possible. Each 
individual case must be judged separately. In Libman's series 
of 50 cases of streptococcemia there were 6 recoveries (11 per cent.); 
of 28 cases of staphylococcemia 8 recovered (nearly 29 per cent.); 
of his 4 pneumococcus cases 1 recovered. Leaving out the few 
pneumococcus cases there would be 86 cases with 16 per cent, 
recoveries. 

In Bertelsmann's series of 48 cases of surgical bacteriemia 21 
recovered, viz., 43 per cent. ; of these there were 28 streptococcus cases 
with 19 recoveries and 13 staphylococcus cases with 4 recoveries. 

In Lenhartz's series of 77 medical cases (including several post- 
partum infections), there were 17 recoveries; of these there were 47 
streptococcus cases with 6 recoveries and 13 staphylococcus cases with 
1 recovery. 

Literature. — F. W. White, "Cultures from the Blood in Septicemia, Pneu- 
monia, Meningitis, and Chronic Diseases," Jour. Exper. Med., vol. iv, p. 425. 
Petruschky, Zeit. f. Hyg., vol. xvii, p. 59. Sittmann, Deutsch. Arch. f. klin. 
Med., vol. liii, p. 323. Canon, Deutsch. Zeit. f. Chir., vol. xxxiii, p. 571; and 
Mitth. aus d. Grenzgeb. d. Med. u. Chir., 1902, vol. x, p. 41. Lenhartz, Munch, 
med. Woch., 1901, Nos. 28 and 29. Libman, Proc. N. Y. Path. Soc, 1903, vol. 
iii, pp. 5 and 57; "On Certain Features of the Growth of Bacteria," etc., Jour 
Med. Research, 1901, vol. vi. Cole, Johns Hopkins Hosp. Bull., 1902, vol. xiii, 
p. 252. Wm. Welch, "Morbid Conditions Caused bv the Bacillus Aerogenes 
Capsulatus," ibid., 1899, vol. x, p. 134. Gwyn, ibid., 1900, vol. xi, p. 185 
(first case); Cole, ibid., 1902, vol. xiii, p. 234 (second case). Hektoen, Jour. 
Amer. Med. Assoc, 1903, vol. xl, No. 11. Jehle, Zeit. f. Heilk., 1901, vol. xxii, 
p. 190. Ewing, Trans. Amer. Assoc. Phys., 1902, vol. xvii, p. 208. D. Rochester, 
Jour. Amer. Med. Assoc, March 2, 1907. C. E. Simon, Meningococcus Septicaemia, 
Johns Hopkins Hosp. Bull., 1907. 

Anthrax. — The bacillus of anthrax, as first pointed out by 
Pollender, Brouell, and Davaine, is frequently met with in the 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 173 



blood in the corresponding disease. As a rule the number is small. 
Smears are stained for five to ten minutes in a mixture of 30 c.c. of a 
concentrated alcoholic solution of methylene blue and 100 c.c. of 
a 1 to 10,000 solution of potassium hydrate; they are then washed for 
five to ten seconds in 0.5 per cent, acetic acid, washed with alcohol 
and dried. Thus stained, the bacilli appear as rods measuring from 
5 to 12 fx in length by 1 p. in breadth, and usually present a segmented 
appearance, the extremities being slightly thickened. Under suit- 
able conditions spore formation takes place. When present in large 
numbers it is not necessary to stain the blood, as the organism can 
then be seen without difficulty in the wet specimen. 

In doubtful cases, in which a 
microscopic examination of the 
blood yields negative results, a 
few cubic centimeters may be 
injected into a mouse or a 
guinea-pig, in the blood of 
which the bacilli will soon be 
found in enormous numbers if 
the disease is anthrax. 

McFadyean has described a 
color reaction of anthrax blood 
which seems to be pathogno- 
monic of the disease. Smears 
are prepared as usual and, 
when air-dry, fixed by heat — 
until the slide has become a 
little too hot to be held against 
the skin. On cooling the specimens are stained for a few seconds 
with a 1 per cent, aqueous solution of methylene blue (medicinal of 
Merck), or with one of Griibler's methylene blues, modified by 
boiling with h per cent, of sodium bicarbonate. After washing 
with distilled water they are dried with filter paper, subsequently 
by heat, and mounted in balsam. Anthrax blood then shows a 
distinct reddish or purplish tone, especially when held against the 
light, while other blood appears pure blue or greenish blue. 

Microscopic examination of the amorphous intercellular material 
shows the same result. 

According to Heim, who has described the same reaction inde- 
pendently of McFadyean, the color change is due to mucin derived 
from the capsules of the bacteria. 

Literature. — Pollender, Casper's Vierteljahrsch. f. gerichtl. u. offentl. Med., 
1855, vol. viii, p. 103. Brauell, Virchow's Archiv, vol. xi, p. 132, and vol. xiv, 
p. 32. Davaine, Compt.-rend. de FAcad. d. Sci., vol. lvii, p. 220. Blumer and 
Young;, Johns Hopkins Hosp. Bull., 1885, p. 127. McFaydean, Jour. Comp. 
Pathol, and Therap., March and December, 1903. Heim, Munch, med. Woch., 
1904, No. 10, 




Fig. 43. — Anthrax bacillus. X 900 diameters. 
Agar culture. (Park.) 



174 THE BLOOD 

Tuberculosis. — In acute tuberculosis tubercle bacilli have repeatedly 
been observed in the blood ; but the search for them is most tedious 
and often in vain. Nevertheless a careful examination of the blood 
is indicated in doubtful cases; but only a positive result is of value. 

According to Liebmann, the tubercle bacilli are most numerous 
in the blood about twenty-four hours after the injection of tuberculin. 
Working in this manner he claims to have obtained positive results 
in 56 cases of 141. As a rule it is best to resort to the animal 
experiment. 

For methods of staining and a description of the tubercle bacillus, 
the reader is referred to the chapter on Sputum. 

Literature. — Liebmann, Berlin, klin. Woch., 1891, p. 393. Kronig, Deutsch. 
med. Woch., 1894, vol. v, p, 42. 

Leprosy. — In leprosy the corresponding bacilli have been found in 
the blood by Mitsuda. 1 Their demonstration, however, is difficult. 

Glanders. — In glanders the specific bacillus is constantly present 
in the blood, and may be demonstrated by staining dried preparations 
for five minutes with a concentrated alcoholic solution of methylene 
blue mixed with an equal volume of a 1 to 10,000 solution of potassium 
hydrate just before using. From this mixture the specimen is passed 
for a second or two into a 1 per cent, solution of acetic acid which has 
been tinged a faint yellow by the addition of a little tropeolin 00 solu- 
tion; it is then decolorized by washing in water containing 2 drops 
of concentrated sulphuric acid and 1 drop of a 5 per cent, solution of 
oxalic acid for each 10 c.c. In specimens thus stained the bacilli 
appear as short rods measuring from 2 [J. to S/J. in length by 0.3/* to 0.4/* 
in breadth, often containing a spore at one end. 

Literature. — Duval, Arch, de med exper., 1896, p. 361. 

Influenza. — The influenza bacillus has been found in the blood 
occasionally, but is more readily demonstrated in the sputum. Jehle 
found it in 22 cases of scarlatina out of 48 that ended fatally, in 
measles 15 times out of 23, and in 5 cases of varicalla out of 9. 
In Hektoen's series, on the other hand, the organism was not found; 
but it is noted that during the year influenza was not especially 
prevalent in Chicago. (For a description of the organism see the 
Sputum.) 

Literature. — Canon, Virchow's Archiv, vol. cxxxi, p. 401. Klein, Baumgar- 
ten's Jahresb., 1893, p. 206. Kiihnau, Zeit. f. Hyg., vol. xxv, p. 492. 

Malta Fever. — In Mediterranean or Malta fever the specific 
organism, the Micrococcus melitensis (Bruce), has been isolated 
from the blood during life. It is said to be present in the peripheral 
blood in all cases during the early stages and in severe febrile relapses. 

1 Folia haematol., vol. i, p. 502. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 175 

In the afebrile intervals and the subsequent cachexial stage it is not 
demonstrable. In no case are the organisms abundant, and for this 
reason the bacteriological findings are rather uncertain. 

Diagnosis is facilitated by the fact that a well-pronounced agglutina- 
tion is obtained with the patient's serum. A positive reaction with a 
dilution of more than 1 to 20, according to Birt and Lamb, may be 
regarded as proof positive of the existence of the disease. As a 
rule agglutination can still be obtained with a dilution of from 1 to 600 
to 1 to 700. It begins about the fifth day of the disease, and gradually 
diminishes in intensity during convalescence, but may persist for a 
year and a half and even longer. 

The organism in question is a coccus, measuring 0.3 it in diam- 
eter, and occurs singly, in pairs, and sometimes in fours. Longer 
chains are not seen. It is motile. It is stained by the usual dyes 
and grows on nutrient agar and in broth. The colonies are usually 
not visible until the third day. At first their color is that of a trans- 
parent amber, while later they are opaque. Liquefaction does not 
occur. 

Literature. — C. Birt and G. Lamb, "Mediterranean Fever," Lancet, Sept. 9, 
1899. Wright and Smith, Brit. Med. Jour., April 10, 1907. Musser and Sailer, 
Phila. Med. Jour., 1898, p. 1408, and 1899. p. 89. R. F. Strong and W. E. 
Musgrave. "The Occurrence of Malta Fever in Manila," Phila. Med. Jour., 1900, 
p. 996. J. J. Curry, "Malta Fever," Jour. Med. Research, July, 1901. 

Bubonic Plague. — In advanced cases of bubonic septicemia 
the specific organism may be found in the blood in small numbers. 
Toward the end of rapidly fatal cases they become more numerous, 
and may then be demonstrable directly with the microscope. Accord- 
ing to Bell 1 the bacilli can be found in all cases and at all stages of 
the disease by using Ross' dehemoglobinizing method (p. 177). 

The organism in question, the Bacillus jpestis (Kitasato, 
Yersin), is a short, thick coccobacillus, with rounded ends, measur- 
ing 1.5 tt to 1.75 p. in length by 0.5 tt to 0.7 tt in breadth. Examined 
in the hanging drop it is devoid of automobility. The polar regions 
are readily stained, while the interpolar area remains colorless. In 
many organisms a capsule can be made out by appropriate methods, 
but it is apparently not a constant feature. Oftentimes the form 
of the organism deviates from the normal. It may thus resemble 
a coccus on the one hand, while on the other it appears more elon- 
gated, and again it is common to meet with distorted and swollen, 
vacuolated forms, which are interpreted as involution or degeneration 
forms. These latter are especially numerous in older cases and old 
cultures. The organism is decolorized by Gram (Fig. 44). 

The blood smears are fixed by immersion in absolute alcohol 
for twenty-five minutes; or they are covered with absolute alcohol 
for about one-half minute, when the alcohol is burned off. For 

1 Brit. Med. Jour. March 5, 1904. 



176 THE BLOOD 

staining purposes, borax methylene blue (a solution of 2 per cent, 
methylene blue in 5 per cent, borax-water) or Loffler's alkaline 
methylene blue may be conveniently employed. In the first in- 
stance we stain for one-half minute, in the second for two to three 
minutes. The polar staining is in this manner quite satisfactory. 

On gelatin and agar containing 2.5 to 3.5 per cent, of salt and in 
bouillon a fairly characteristic growth results. In the case of the 
agar involution forms are obtained, among which long, slender 
bacilli, which are segmented and present a vacuolated appearance, 
are especially noteworthy. In this state they stain quite badly and 
have lost a certain degree of their virulence. In bouillon the or- 
ganism often forms long chains of well-rounded bodies which are 



a 










' ■-*■• ic . ,.... *•»■■"' 
Fig. 44. — Plague bacilli from agar culture. X 1100 diameters. (Park.) 

quite similar to a coccus. During its growth in bouillon it forms 
flakes or flocculi, which rapidly sink to the bottom of the tube, 
leaving the liquid clear above. Stalactite or stalagmite formations 
may also be seen, starting from the walls of the tubes or from sus- 
pended droplets of oil or butter. Colonies on gelatin about thirty- 
six hours old are warty, strongly refractive formations, which often 
present a delicate, irregularly indented margin. Even after twenty- 
four hours one can obtain smears in which 50 to 100 bacilli are 
grouped in little colonies of irregular form, while examination of the 
plates with a magnifying power of 60 diameters reveals scarcely any 
growth. The organism does not liquefy gelatin. The optimum 
temperature for growths is between 25° and 30° C. 

Literature. — For Kitasato's report see Annual Rep. of the U. S. Marine- 
Hospital Service for 1894; W. Wyman, Bubonic Plague; U. S. Treasury Dept., 
1900. Kossel u. Overbeck, Arb. aus. d. Kais. Gesundheitsamt., 1901, vol. xviii. 



ANIMAL PARASITES 177 



ANIMAL PARASITES. 



Malaria. — Malarial fever is referable to infection with a specific 
protozoan parasite belonging to the class of hematozoa, representa- 
tives of which are found in the blood of various animals, such as the 
rat, frog, turtle, carp, various birds, etc. Three varieties are known 
to occur in the blood of man, viz., the parasite of tertian, quartan, 
and estivo-autumnal fever. The life history of these organisms is 
now well understood, and it is known that in addition to the intra- 
corporeal cycle of development which takes place in the human body 
there is yet another, an extracorporeal cycle, which occurs in certain 
mosquitoes of the genus Anopheles. Infection occurs through the 
bites of such mosquitoes, which themselves have been infected by 
sucking the blood of malarial patients. This has been abundantly 
demonstrated by Ross, Manson, Grassi, and others, and may be 
regarded as an established fact. 

Method of Examination. — When the patient is directly available at 
the laboratory, or if a few hours only need elapse before the examina- 
tion is made, wet mounts may be used, which are best ringed with a 
little vaselin or paraffin, if they cannot be examined at once. Other- 
wise dry mounts are prepared and stained with the eosinate of methy- 
lene blue, or one of the Romanowsky dyes, such as Hastings', Wright's, 
Giemsa's, etc. (See Plate X.) With the Romanowsky mixtures, 
which all contain methylene azure, the chromatin (nuclear) granules 
are shown. 

It is best to procure specimens shortly before an attack, as adult 
forms are then obtained ; immediately after an attack is not the proper 
time to hunt for parasites. 

In cases in which but few organisms are expected Ross has suggested 
the advisability of spreading thick blood specimens and extracting 
the hemoglobin before staining. The search for the youngest forms 
of the estivo-autumnal parasite especially is much facilitated in this 
manner. Ruge endorses this method in the following modification, 
but points out that the specimens are by no means beautiful. A 
large drop of blood (about 20 cb. mm.) is spread over a surface measur- 
ing about 18 square millimeters. The air-dried preparation is then 
placed for a few minutes in a 5 per cent, solution of formalin, 1 to which 
0.5 to 1 per cent, of acetic acid has been added. In this manner the 
hemoglobin is all extracted, while at the same time the blood film 
is fixed; so that it can now be washed without fear of ruining the 
preparation. This is then stained either according to one of the 
modifications of the Romanowsky method or with the eosinate 

1 This solution would contain 2 per cent, of formaldehyde gas, as the commercial 
formalin is about a 40 per cent, solution. 

12 



178 THE BLOOD 

of methylene blue. Huge further advises that specimens stained 
according to the Romanowsky method be subsequently stained with 
Manson's solution, 1 in order to render the smallest and medium- 
sized ring forms more readily visible, as their affinity for the dye is 
somewhat impaired by the fixation in formalin. My own experience 
with this method has been very satisfactory. 

Plasencia suggests the following method: Fixation in 0.5 per cent, 
formalin and absolute alcohol (equal parts); rapid drying in the air 
and washing in distilled water. The specimens are then stained 
with a mixture composed of 80 c.c. of a saturated aqueous solution 
of toluidin blue and 60 c.c. of a 1 per cent, aqueous solution of eosin. 
After washing in water they are dried and examined as usual. Pla- 
senica regards this stain as better than Manson's. 

The Parasite. — The following forms of the parasite may be found 
in the blood: 

1. Hyaline Non-pigmented Intracellular Bodies. — These 
apparently represent the earliest stage in the development of the 
parasite, and are found in all forms of malarial fever; they are espe- 
cially abundant during the latter part of the paroxysm or immedi- 
ately thereafter. At first sight they may be mistaken for vacuoles, 
but upon closer examination it will be found that they exhibit dis- 
tinct movements of an ameboid character, and may thus easily be 
recognized with a little experience. 

The rapidity with which these changes in form occur in the tertian 
type of ague is most astonishing, and sketches of any one phase can 
often, indeed, be made only from memory; in quartan fever the 
movements are much slower and far less extensive. 

In the irregular fever of the estivo-autumnal form ameboid move- 
ments may likewise be observed, but more commonly the para- 
site assumes a ring-like appearance, and does not throw out distinct 
pseudopodia. If these forms are carefully observed, however, it will 
be found that they are not absolutely quiescent, but alternately ex- 
pand and contract. 

In tertian fever the organism (Plate VIII) is pale and indistinct, 
while in quartan fever it is sharply outlined and somewhat refractive 
(Plate IX, Fig. 2). In the estivo-autumnal form the organism is 
usually much smaller than in the tertian type, and the ring-like bodies 
frequently present at some point in their interior a distinctly shaded 
aspect which closely resembles the darker portion in the centre of a 
normal corpuscle (Plate IX, Fig. 1). It is thus possible, even at this 
stage in the development of the parasite, to distinguish between fever 
of the tertian, quartan, and estivo-autumnal type. 

1 This is an aqueous solution of borax (5 per cent.) and methylene blue (2 per 
cent.). The blood films are stained with this solution for about thirty seconds; 
they are then washed in water, dried with filter paper, and afterward b}^ gently 
warming them over the flame. 



PLATE VIII 














LSdirmdt fecit 



The Parasite of Tertian Fever. 



1, normal red corpuscle ; 2 to 4, non-pigmented stage of the organism, showing ameboid move- 
ments ; 5 to 7, progressive pigmentation and growth; 8 to 11, process of segmentation; 12, young 
forms ; 13, large extracellular organism ; 14, mode of formation of extra-cellular body ; 15, small 
fragmented extra-cellular organism ; 16, flagellated body and free flagella. Unstained specimen. 
(Personal observation.) 



ANIMAL PARASITES 179 

2. Pigmented Intracellular Organisms. — These represent a 
later stage in the development of the parasite, and, like the non- 
pigmented intracellular bodies, are met with in all types of malarial 
fever. Their appearance, however, differs considerably in the vari- 
ous forms. In tertian fever minute granules of a reddish-brown 
color appear in the bodies of the organism soon after the paroxysm. 
These gradually increase in number, while the invaded corpuscles 
proportionately become paler and paler, until finally only an indis- 
tinct, shell-like outline can be discerned. In fresh specimens the 
granules, which often assume the form of little rods, resembling 
bacteria, exhibit most active molecular movements, attracting atten- 
tion at once. The body of the parasite, which during its develop- 
ment has increased gradually in size, is probably hyaline, and may 
still be seen to undergo ameboid movements. These are not nearly 
so active, however, as in the non-pigmented stage. The move- 
ments, moreover, cannot be followed so readily, owing to the pres- 
ence of the granules. At first sight these appear to be scattered 
in small collections throughout the red corpuscles, and the impression 
may be gained that several organisms are present at the same time. 
Upon closer investigation, however, it will be seen that this is only 
apparently the case, and that the granules are confined to the bulb- 
ous extremities of the pseudopodia of a single parasite. Before 
the end of forty-eight hours the organism has filled out the entire 
red corpuscle, which at the same time has attained a larger size 
than normal. The ameboid movements become less and less marked, 
and the pigment granules, which may still be quite active, tend to 
collect about the periphery (Plate VIII). 

In quartan fever pigmented intracellular bodies likewise appear 
soon after the paroxysm. The individual granules, however, are 
somewhat larger, of more irregular size, and darker in color than 
those seen in the tertian type (Plate IX, Fig. 2). Instead of ex- 
hibiting active molecular movements, moreover, they are almost 
entirely quiescent, and usually are grouped along the periphery of 
the organism. While ameboid movements can at first be observed, 
these become less and less marked, until finally, at the end of from 
sixty-four to seventy-two houis, they cease. The organism then 
presents a round or ovoid form, but does not fill the red corpuscle 
entirely. It is curious to note that in this form of ague the red 
corpuscles do not become decolorized, but rather darker than normally 
and at times specimens may be seen which present a distinctly green- 
ish or brassy appearance. When the parasite has become fully 
developed the corpuscle is smaller than normally, and, on staining, 
it may be seen that the organism still is surrounded by a narrow 
zone of corpuscular protoplasm even when this is not apparent in 
unstained preparations. 

The pigmented intracellular bodies which may be found in estivo- 



180 THE BLOOD 

autumnal fever (Plate IX, Fig. 1) can readily be distinguished 
from those observed in tertian and quartan ague. As in those 
types, pigment granules also appear after the paroxysm; they are 
never numerous, however, and often only one or two minute dark 
granules can be detected near the periphery. The organism, even 
in the later stages of its development, scarcely ever occupies much 
more than one-third of the corpuscles. Usually the granules exhibit 
scarcely any movements. As in the quartan type of ague, decolor- 
ization of the red corpuscles does not occur, and here, as there, a 
greenish, brassy appearance often is observed. 

At the beginning and during the paroxysm forms are at times 
seen in which the few pigment granules that may be present have 
gathered in the centre of the parasite and formed a solid clump. 
From the facts that these are observed only during the paroxysm, 
and that central blocks of pigment are found only during the stage 
of segmentation (see below) in tertian and quartan ague, Thayer 
and others conclude that these bodies are presegmenting forms of 
the parasite. This belief is strengthened by the observation that 
pigment-bearing leukocytes are then also seen, which in the other 
types of fever likewise are found only at this time. 

3. Segmenting Bodies. — In cases of tertian and quartan fever the 
process of segmentation may be observed directly under the micro- 
scope, if specimens of blood are obtained just prior to or during the 
chill. In tertian fever organisms will then be seen in which the de- 
struction of the red corpuscles has advanced to a stage in which it is 
only possible to make out a pale contour of the original host. The 
parasite itself has gradually assumed a granular appearance, and the 
pigment granules, which until then have exhibited pronounced mo- 
lecular movements, now become quiescent, larger and rounder, and 
show a distinct tendency to collect in the centre of the body. Here 
they form a roundish mass in which the individual components can 
scarcely be made out. While this change in the position of the pig- 
ment is taking place, beginning segmentation of the surrounding 
granular protoplasm will be observed. This at first is most marked 
at the periphery, from which delicate striae will gradually be seen to 
extend toward the central mass, dividing up the protoplasm into a 
number of oval bodies which closely resemble the petals of a flower 
(Plate VIII). Still later these bodies, which in reality are the 
sporules (merozoites) of the parasite, will be found scattered in 
an irregular manner throughout the interior of the organism. The 
apparent envelope then disappears, and the sporules, which in tertian 
fever usually number from fifteen to twenty, lie free in the blood. 
Quite frequently, also, a sudden expulsion of the little bodies is 
observed and the impression gained as though the envelope had been 
burst asunder. Upon closer inspection, even at the petal stage, it will 
be seen that almost every sporule presents a tiny dot in its interior, 



PLATE IX 







a 









ft. 





L Sclvnidt feat 

The Parasite of Estivo-autumnal Fever. 

1, normal red corpuscle; 2 to 10, gradual growth of the organism ; 11 and 12, segmenting bodies ; 
13, young forms ; 14 to 22, crescents, ovoids and spherical bodies, with and without bib ; 23, flagellated 
body. Unstained specimen. (Personal observation.) 





Q 







LScfunuit fecit 



The Parasite of Quartan Fever. 

1, normal red corpuscle ; 2 to 6, gradual growth of the organism ; 7, pigmented extracellular 
body ; 8, segmenting body ; 9, young forms ; 10, vacuolated extracellular body ; 11, flagellated form. 
Unstained specimen. (Personal observation.) 



ANIMAL PARASITES 181 

which may at first sight be mistaken for a pigment granule, but 
which in all probability is a nucleus. After the expulsion of the 
sporules these are frequently seen to move about in an active manner, 
but sooner or later they come to rest. 

While the progress of segmentation is very frequently observed to 
proceed in the manner described, this is not invariably the case. It 
may thus happen that segmentation occurs before the pigment 
granules have had time to gather at the centre, or that the parasitic 
protoplasm breaks up into sporules directly without the intervention 
of the petal stage. In every case, however, the formation of sporules 
is associated directly with the occurrence of a paroxysm and repre- 
sents the asexual type of reproduction of the parasite (schizogony). 

The sporules, unless destroyed by leukocytes, in turn invade new 
corpuscles, cause their destruction, and become segmented, thus 
giving rise to a new generation. As the process of segmentation 
coincides in time with the occurrence of the chill, it is apparent that 
the interval elapsing between two consecutive chills — i. e., the type 
of the ague — depends upon the rapidity with which the organisms 
arrive at maturity. 

In quartan ague segmentation differs somewhat from that observed 
in the tertian form. It will here be observed that the pigment 
granules, which have gathered along the periphery of the organism, 
as the parasite approaches maturity become arranged in a stellate 
manner, and apparently reach the centre through definite protoplasmic 
channels. Here they form a dense clump, and while the protoplasm 
assumes a finely granular appearance, segmentation proper begins 
and proceeds as in the tertian form. The number of segments, 
however, is smaller, varying between six and twelve. The entire 
segmenting body, moreover, is smaller than in the tertian form, and 
the segments are arranged in a more symmetrical manner. Here, 
indeed, the most perfect rosettes are observed (Plate IX, Fig. 2). 

In estivo-autumnal fever segmenting bodies are only exception- 
ally seen in the peripheral blood, and it appears that the process of 
reproduction occurs principally in the spleen. The segments, as a 
rule, number from ten to twenty. The segmenting body itself, 
however, is much smaller than in either the tertian or quartan form, 
and it is not possible to distinguish any remains of the original host. 

4. Crescents, O voids, and Spheroids (Plate IX, Fig. 1) — 
These are observed only in cases of estivo-autumnal fever when 
this has persisted for at least a week. At first sight they apparently 
bear no relation to the other forms which have been described, but 
it is known that they are derived directly from the pigmented intra- 
cellular forms. Specimens may thus be met with in which cres- 
centic bodies are found in the interior of red corpuscles that have 
lost but little of their original color.. Such observations, however, 
are not common. The typical crescents which are usually seen are 



182 



THE BLOOD 



highly refractive bodies, somewhat larger than a red corpuscle, 
measuring from 7 /J. to 9 /J. in length by 2 p. in breadth. Their ex- 
tremities are usually rounded off and joined by a delicate, curved line 
bridging over their concave border. This is supposed to represent 
the remains of the original host. At other times this hood-like 
appendage is found along the convex border. The little pigment 
granules and rods, which are always found in the interior of the 
crescents, are generally collected about the centre of the body, but 
they are occasionally also seen in one of the horns. While usually 
quiescent, a migration of some of the granules toward one extremity 
and back to the central mass may be observed. The ovoid and 




Culex. 



Anopheles. 



Anopheles. 
Fig. 45. — (From Doflein.) 



Culex. 



spherical bodies, which are usually smaller than the crescents, exhibit 
the same general features and often are provided likewise with a 
little hood. It is now known that the spherical bodies develop from 
the ovoids, and these again from the crescents. 

5. Extracellular Pigmented Bodies or Gametes. — In tertian 
and quartan ague some of the pigmented intracellular bodies, instead 
of undergoing segmentation when they have arrived at maturity, 
leave their hosts and appear as such in the blood. Some of them 



PLATE X. 



.A 









W£z~ ~* 



%>• 











H 







i. 



Malarial Organisms. 

a, young estivo-autumnal ring bodies; b, young tertian form; c, tertian parasites in various 
stages of development; d, segmenting organism; e, estivo-autumnal crescents; /, large mononucleai 
leukocyte carrying pigment from ingested malarial organisms. Stained with Wright's stain. 



ANIMAL PARASITES 183 

at the same time increase considerably in size, and in the tertian 
form may become as large as a polynuclear leukocyte (Plate VIII). 
The pigment granules, moreover, exhibit an activity in their move- 
ments which is most astonishing and never observed under other 
conditions. The outline of the parasite is then usually irregular 
and quite indistinct. Upon careful observation it will be seen 
that in some of these bodies the movements of the granules after a 
while become less and less marked, and finally cease, while the body 
of the parasite itself becomes still more irregular in outline. This 
appearance is undoubtedly referable to the death of the organism. 
In others a gradual fragmentation is observed, small particles of the 
pigmented mother-substance being cut off from the parent form. 
It is thus quite common to see the original parasite break up into 
four or five smaller bodies, in which the movements of the pigment 
granules persist for some time. Sooner or later, however, even these 
cease, the outlines of the bodies become more and more indis- 
tinct, and death occurs. In still others the formation of vacu- 
oles may be observed, the pigment granules at the same time 
becoming quiescent. This process is likewise regarded as one of 
degeneration. Most interesting, however, is the fact that flagel- 
lation may occur in some of these extracellular forms. This may 
sometimes be hastened in the wet specimen by gently breathing 
upon the slide so as to form a thin film of moisture It will then 
be observed that the pigment granules which exhibit a most sur- 
prising activity tend to collect near the centre of the organism, while 
at the same time curious undulating movements may be made out 
along its contours. Suddenly one or more (one to six) slender fila- 
ments will be seen to protrude from as many points on the periphery, 
presenting minute enlargements here and there in their course (Plate 
IX) (polymites). The length of these filaments, or flagella, as 
they are termed, varies considerably. As a rule, it does not exceed 
the diameter of from five to eight red corpuscles, but longer speci- 
mens are at times observed. With these flagella the organism makes 
most active whipping movements, scattering the red corpuscles to 
the right and left. Attention is, indeed, usually first drawn to the 
presence of these bodies by the disturbance which they cause in the 
field of vision. Occasionally one of the flagella may be seen to become 
detached from the body of the parasite and to move rapidly about 
among the corpuscles in a snake-like manner. In microscopic speci- 
mens they gradually come to a rest and often curl into a spiral. 

Beyond the fact that the flagellate organisms in tertian fever are 
larger than in the quartan form, no special points of difference exist 
(Plate IX, Fig. 2). 

In estivo-autumnal fever similar changes may be observed. The 
flagellate forms are here direct derivatives of the crescents, which have 
changed to ovoids and these to spheroids. The flaggellates, as in 



184 



THE BLOOD 



quartan fever, are smaller than those observed in the tertian form 
(Plate IX, Fig. 1). 

The significance of the flagellate organisms is now well under- 
stood. They represent the male element in the sexual reproduction 
of the malarial parasite (microgametocytes) and the beginning of a 
cycle of development, which takes place outside of the human body, 
in the bodies of mosquitoes of the species Anopheles. The beginning 
of this cycle was first observed by MacCallum in the blood of infected 
crows. He here discovered that when one of the flagella (micro- 
gametes) broke loose it almost always sought out another full- 
grown form of the parasite which had not undergone segmentation, 
and penetrated this, just as the spermatozoon penetrates the ovum. 



Schema of double cycle. 

L. B. Gohihorn, fee. 
190k. 





"Oval rest-body in 
wall of stomach. 

( \ookinet which penetrates 
\G») lining of stomach-wall. 

Mosquito 






Flagellation 
in stomach 
of mosquito. 

Inner circle-asexual reproduction; moist- Infection of man through gastro-intestinal and 

chamber observation shows no flagellation. respiratory tract, the infected mosquito dying 

Outer circle-formation of (sexuah gametes; in water, drying in air or sucking pi ant -juices, 

moist-chamber observation shows flagellation. infecting fresh vegetables (theoretical) 

Fig. 46. — Illustrating cycle of development. (Park.) 

Subsequently he observed the same process in the blood of the human 
being, which has since been confirmed by others. The female 
cells are somewhat larger than the male cells and termed macroga- 
metes. The further development (sporulation) of the fertilized forms 
(ookinetes), however, does not take place in the human blood, but in 
the mosquitoes. The fertilized organism penetrates the stomach 
wall of the insect and here gives rise to the formation of little cysts 
(oocysts) in which after about seven days numerous irregular, rounded, 
ray-like strite appear. After a time the capsules of the cysts burst 
and the delicate, thread-like bodies (the sporozoites) are set free in the 
body cavity of the mosquito, and shortly after appear in the salivary 
glands. These bodies represent the young parasites, which result 
from the sexual reproduction of the adult organism. If at this stage 



ANIMAL PARASITES 185 

of their development the infected mosquito is allowed to bite a human 
being malarial infection results, with the appearance in the blood of 
the hyaline forms already described. 

From the above description it will be seen that three forms of 
the malarial parasite may be found in the blood, viz., the parasite of 
tertian, quartan, and estivo-autumnal fever, and it has been shown 
that these forms may readily be distinguished from each other. In 
tertian and quartan fever several groups of the same organism may 
be present at one time, and as the process of segmentation coincides 
with the occurrence of a paroxysm it will readily be seen that the 



&! :\m\i\*. 



Fig. 47. — Ookinetes of pernicious parasites in the stomach of Anopheles maculipennis 
thirty-two hours after having been sucked in. (Grassi.) 




Fig. 48. — Transverse section of the stomach of an anopheles, with cysts of pernicious 
parasites. (Grassi.) 

number of paroxysms within a given time depends upon the number 
of groups which may be present in the blood. If a double infection 
with the tertian parasite has occurred, one group of organisms may 
thus have just reached the segmenting stage, while the second group 
has attained only a twenty-four hours' growth, the result being that 
maturity is reached by the two groups on successive days. Quotidian 
fever is then the result. Should still other groups be present, the 
clinical picture will accordingly become more complicated. In 
quartan ague, similarly, double quartan fever will occur if two groups 
are present, and triple quartan fever if three groups are present at one 
time. Mixed infections, further, are also possible. 

Pigmented Leukocytes. — In conclusion, it may not be out of 
place to refer to the presence of pigment-bearing leukocytes in the 



186 



THE BLOOD 



blood of malarial patients. (See Plate X.) These are quite constantly 
met with during the paroxysm, and it is indeed often possible to 




Fig. 49. — Four stages of sporulation of malarial parasites from Anopheles maculipermis, 
strongly magnified: a-c, the pernicious parasite; a, four to four and a half days after ingestion; 
b and c, five to six days after ingestion; d, tertian parasite, eight days after ingestion. (Grassi.) 





Fig. 50 — Section through a tubule of the salivary gland of an anopheles, with sporozoites of the 
pernicious parasites; above an isolated sporo'zoite with higher magnification. (Grassi.) 

observe the process of phagocytosis directly under the microscope. 
The forms which are taken up are the small, fragmented, extra- 
cellular forms, the flagellate bodies, segmenting bodies, and free 



ANIMAL PARASITES 187 

pigment clumps. In every ease where pigment-bearing leukocytes 
are observed, malarial fever should be suspected and a careful 
examination made, as a melanemia only occurs in this disease, in 
relapsing fever, and in connection with the rare melanotic tumors, 
in which not only leukocytes containing melanin may occur in large 
numbers, but also masses of pigment floating free in the blood. 

Literature. — A. Laveran. Nature parasitaire des accidents de l'impaludisme, 
Description d'un nouveau parasite, Paris, 1881. P. Manson, Tropical Diseases, 
Cassell & Co., London, 1900, p. 1. For a full account of the literature, see the 
monograph by W. S. Thayer and J.jHewetson/' The Malarial Fevers of Baltimore," 
Johns Hopkins Hosp. Rep., vol. v. On recent advances in our knowledge con- 
cerning the etiology of malarial fever, see W. S. Thayer, Phila. Med. Jour., 1900. 
p. 1046, where a full account of the literature is given. T. B. Futcher. " A Critical 
Summary of Recent Literature concerning the Mosquito as an Agent in the 
Transmission of Malaria," Amer. Jour. Med. Sci., 1899. p. 318. W. S. MacCallum, 
''On the Hematozoon Infection of Birds," Jour. Exper. Med., vol. hi. p. 117. 
E. L. Opie, " On the Hematozoon of Birds," ibid., p. 79. F. Grohe, " Zur Gesch 
d. Melanaemie " Yirchows Archiv, 1861, vol. xx, 306. 




Fig. 51. — Trypanosoma gambiense (sleeping sickness) in blood of a rat. Two t5*pes are 
shown; the broad, pale form (female?) is dividing. Magnification 1500 times. MacXeal'? 
stain. (From Xovy.) 

Trypanosomiasis. — The first authentic report concerning the occur- 
rence of trypanosomiasis in man was made by Dutton in 1902, while 
in animals their occasional presence had long been recognized (frogs, 
rats, dogs, groundhogs, etc.). In tropical regions certain species are 
pathogenic for certain domestic animals. The tse-tse fly disease or 
Xagana of Africa, the Surra disease of Asia, and the mal de caderas 
of South America are all referable to infection with trypanosomes 
(observed in the horse, the African buffalo, the ox, the donkey, 
mule, antelope, camels, and elephants). Especially interesting is the 
observation of Castellani and Bruce of the association of trypanoso- 



188 THE BLOOD 

miasis with a certain symptom-complex, of which the so-called sleep- 
ing sickness is one of the possible manifestations. Bruce could 
demonstrate the organism in the blood of 12 out of 13 cases, and 
in the cerebrospinal fluid in all of 38 cases. The findings of these 
earlier observers have since been abundantly confirmed, and it is now 
generally conceded that the disease in question is referable to infection 
with trypanosomes. 

The Trypanosoma gambiense (Dutton) is from 8 to 25 fi long, and 
from 2 to 2.8 fJ. broad. It is provided with an undulating mem- 
brane and a flagellum, which starts from a centrosome or micronucleus 
lying in the posterior end of the animal, and projects somewhat 




Fig. 52.— Trypanosoma gambiense from same preparation as preceding, showing the usual 
form; some cells in process of division. Magnification 1500 times. (From Novy.) 

beyond the anterior end. (See Figs. 51 and 52.) There is an oval 
nucleus which is centrally located and is made up of chromatin 
granules. 

In the wet preparation the organism exhibits slow spiral move- 
ments. It is found free in the blood plasma, but may also be seen 
in the interior of leukocytes, which latter manifestly destroy the 
organisms exactly as the malarial parasites. In dry specimens the 
trypanosomes can be readily stained with any basic dyes; with the 
Romanowsky stain or one of its modifications it is stained like the 
malarial organism. Levaditi 1 recommends the following method as 
especially valuable: Fixation in absolute alcohol and ether for five 
minutes; primary staining for two minutes with a saturated solution 
of Bismarck brown, followed by washing and counterstaining with 

1 Soc. de biol., November 23, 1903. 



ANIMAL PARASITES 189 

Unna's polychrome blue (diluted one-half with water) for two minutes. 
The specimens are rinsed in water, dried over a flame, and examined 
as usual. 

The number of organisms in a blood preparation is rarely large; 
as a rule, not more than 3 to 8 are found to a cover-slip. During 
apyrexia they are not seen. 

Infection in man probably occurs through a biting fly — the Glossina 
palpalis, which supposedly transmits the disease in a purely mechani- 
cal way. 

Novy and McNeal succeeded in cultivating the trypanosoma of 
Bruce in the water of condensation from a medium of agar mixed 
with defibrinated rabbit's blood (1 to 1) at 25° C, and the rat trypano- 
some (Trypanosoma lewisi) in a similar medium containing 1 part 
of blood for 2, 5, or even 10 parts of agar. 

Literature. — Dutton, Thompson- Yates Laboratory Rep.. 1902, vol. iv, part 
ii, p. 455; and Brit. Med. Jour., 1903, vol. i, p. 304. Castellani and Bruce, ibid., 
pp. 1218 and 1431; Jour. Trop. Med., 1903, p. 167. Novy and McNeal, Journ. 
Amer, Med. Assoc, November 21, 1903. Novy, ibid., Jan. 5, 1907. 

Relapsing Fever. — Relapsing fever is characterized by the 
presence in the blood, and here only, of spirochetes which bear the 
name of their discoverer, Obermeier. In order to search for the 
organisms no special precautions are necessary. After having 
cleansed the finger a drop of blood is mounted on a very thin cover- 
glass. This is inverted directly upon a slide, when the specimen 
is ready for examination; an oil-immersion lens is not required. 
Attention is drawn to the presence of the organisms by disturbances 
which are noticeable among the red corpuscles, and upon careful 
examination it will be seen that these are caused by the wriggling 
movements of the spirochetes. The Spirochsetse Obermeieri are 
long, slender filaments, measuring from 36 ft to 40 fJ- in length 
by 0.3 fJ. to 0.5 fi in breadth, and present from eight to twelve in- 
curvations of equal size with tapering extremities. These two last 
characteristics serve to distinguish this species from that described 
by Ehrenberg, in which the radius of the incurvations is not the 
same in all, and in which the extremities do not taper (Fig. 53). 

The number of spirilla which may be found in a drop of blood 
varies, being greater during the access of the fever, when twenty, or 
even more, may be observed in the field of the microscope. They 
occur singly or in bunches of from four to twenty. In the quiescent 
stage they are arranged sometimes in the form of rings or of the figure 
8. After the crisis they seem to disappear entirely, and their pres- 
ence during an afebrile period may therefore be regarded as indi- 
cating a pseudocrisis. During the afebrile periods small, bright, 
round bodies have been described in the blood, which according to 
some are spores, but according to others represent merely debris 
of the spirilla. 



190 THE BLOOD 

Culture experiments have not been very satisfactory, although 
Koch observed an increase in their number at a temperature of from 
10° to 11° C. 

Koch has shown that in African relapsing fever, which is likewise 
due to a spirocheta, infection occurs through the bite of a certain 
tick, Ornithodorus moubata, which acts as intermediary host in the 
development of the organism. 




Fig. 53. — Spirochaetae Obermeieri; blood smear, x 1000 diam. (From 
Itzerott and Niemann.) 

The tick fever of the Congo Free State is apparently identical with 
the African recurrens described by Koch. Infection likewise occurs 
through the bite of infected ticks, Ornithodorus moubata. 

Hodlmoser has shown that the blood of recurrens is spirilla 
agglutinating. But as the culture of the organisms is practically 
not possible, the blood of a second case must be available for the test. 

Literature. — Heidenreich. Untersuch. iiber d. Parasit. d. Riickfallstyphus, 
Berlin, 1877. Moczutkowsky, Deutsch. Arch. f. klin. Med., vol. xxiv, p. 80, and 
vol. xxx, p. 165. Blisener, Inaug. Diss., Berlin, 1873. Engel, Berlin, klin. 
Woch., 1873, p. 409. J. E. Dutton and J. L. Todd, Brit. Med. Jour., November 
11, 1905. 76 Versammlung d. Nat. u. Azt., Breslau, 1904. 

Typhus Fever. — According to Gottschalk 1 a protozoon, closely 
related to Pyroplasma bigonicum, which he terms Apiosoma, can be 
demonstrated in the blood of typhus fever. He claims to have found 
sporulation cysts and flagellated forms. Infection according to 
Gottschalk may occur through bedbugs. 

Tropical Splenomegaly (Kala-azar). — Through the researches 
of Donovan, Leishman, and Ross especially it has been established 
that in tropical splenomegaly (cachexial fever, Kala-azar) parasites 
may be demonstrated in the blood which are probably etiologically 
connected with the pathological condition. The organism in question 
has been termed the Leishmania Donovani (Leishman-Donovan 
body, Cunningham-Leishman-Donovan body). It represents a stage 

1 Deutsch. med. Woch., 1903, No. 19. 



LATE XI 



,% 
V 



» 



# 



S§5 



Leishmania-Donovani. 

if leukocytes undergoing dissolution. (Stained with Leishman's stain.) 



ANIMAL PARASITES 191 

in the development of a trypanosome, as was suggested by Rogers and 
as has since been shown by cultural experiments by Leishman and 
Statham. 

In the peripheral blood the organisms are rarely found and only 
when the temperature is high. Splenic puncture gives the best 
results. Donovan suggests that it is well to keep the patient flat on 
the back for twenty-four hours after the operation and to give a 
dose of calcium chloride immediately after and twice again at intervals 
of three hours (to prevent hemorrhage). The parasites are princi- 
pally met with in large mononuclear cells. The typical forms are 
oval or circular with a well-marked contour (Plate XI). There is a 
deeply staining nucleus lying against the capsule and a deeply stain- 
ing, rod-like centrosome. They may occur singly or in pairs or in 
zooglcea masses. They are readily stained with any one of the 
methylene -azure mixtures (Hastings, Giemsa, Leishman, etc.). 

Literature.— R. Ross, Brit. Med. Jour., July 9, 1904. L. Rogers, Lancet, 
July 23, 1904. Leishman and others, Discussion, Brit. Med., Jour., September 
17, 1904. Leishman and Statham, Jour. Royal Army Med. Corps, March, 1905. 

Syphilis. — The Spirocheta pallida (Treponema pallidum) has been 
demonstrated in the blood during life. Under ordinary circumstances, 
however, its search is here not likely to be attended by success. For 
diagnostic purposes it should be looked for in scrapings from chancres, 
papules, condylomas, in the aspirated juice of enlarged lymph glands, 
etc. (For a description of the organism see Examination of Syphilitic 
Material.) 

Spotted Fever. — In the so-called spotted fever, which occurs in 
Montana, Nevada, Oregon, etc., an intracorpuscular ameboid, non- 
pigmented organism has been described by Wilson and Chowning, 
as also by Anderson, which they regard as the cause of the disease. 
They term this the Pyroplasma hominis. Infection supposedly 
takes place through ticks belonging to the species Dermacentor 
reticulatus. 

I have studied the blood in several cases which were placed at 
my disposal by Drs. McCalla, Maxey, and Pease, but was unable 
to find such structures. Craig and Stiles express themselves in a 
similar manner. 

Literature. — Wilson and Chowning, Jour. Amer. Med., Assoc, 1902, vol 
xxxix, p. 131. J. F. Anderson, Amer. Med., 1903, vol. vi, p. 506. Craig, Amer. 
Med., December 10, 1904. 

Filariasis. — According to Manson, the embryos of at least four, 
and possibly five and even more distinct species of nematodes may be 
found in the blood of man. These various blood worms Manson 
designates as the Filaria nocturna, Filaria diurna, Filaria perstans, 
Filaria demarquaii, Filaria ozzardi (a doubtful species), and a sixth, 
which may or may not be connected with one of the two last, the 



192 THE BLOOD 

Filaria magelhsesi. Two of these at least are of pathological import, 
viz., the Filaria nocturna and the Filaria perstans. 

Filaria Nocturna (Manson): syn., Filaria sanguinis hominis 
(Lewis). This filaria is the embryo form of the Filaria Bancrofti 
(Cobbold), which inhabits the lymphatics and is unquestionably the 
cause of endemic chyluria, of various forms of lymphatic varix, of 
tropical elephantiasis arabum, and possibly also of other obscure 
tropical diseases. The organism in question is widely distributed. 
It is indigenous in almost all tropical and subtropical countries as 
far north as Spain in Europe and Charleston in the United States, 
and as far south as Brisbane in Australia. It is very common in 
Cochin and in some of the South Sea Islands, where one-third and 
one-half of the population, respectively, appear to be infected. 

In the following description of both parent and embryo form I 
quote largely from Manson's account of the parasite in his admirable 
manual of tropical diseases. 



■° f° 


°% 




% ! 

. o i 





Fig. 54. — Filaria sanguinis. 

The parent filarias are hair-like, transparent worms measuring from 
7.5 to 10 cm. in length. The sexes live together, often inextricably 
coiled about each other. Sometimes they are enclosed, coiled several 
in a bunch, and tightly packed in little cyst-like dilatations of the 
distal lymphatics; sometimes they lie more loosely in lymphatic 
varices; sometimes they inhabit the large lymphatic trunks between 
the glands, the glands themselves, and probably not infrequently the 
thoracic duct. The female is the larger; there are two uterine tubes 
which occupy the greater part of the body, and which are filled 
with ova in various stages of development. The vagina opens near 
the mouth; the anus just in advance of the tip of the tail. The 
cuticle is smooth and without markings. In both sexes the mouth 
end tapers slightly; it is clubbed and simple. The male is charac- 
terized by its marked disposition to curve. The cloaca gives exit to 
two slender, unequal spicules. 



ANIMAL PARASITES 193 

In the wet preparations the Filaria nocturna appears as a trans- 
parent, colorless little worm, which wriggles about most actively, 
constantly agitating and displacing the corpuscles in its vicinity. It 
will be noticed, however, that the animal does not propel itself 
through the drop of blood, but remains stationary. At first the 
movements are so active that it is impossible to make out any ana- 
tomical details; after a number of hours, however, the movements 
become more sluggish, and it is then possible to study the worm 
with more ease. It measures about 0.31 mm. in length by 0.007 to 
0.008 mm. in width. With the higher power it will be seen that the 
entire worm is enclosed in a delicate envelope, in which it moves 
backward and forward, the sheath being much larger than the worm 
(Fig. 54) . It is owing to the presence of this sheath that active loco- 
motion on the part of the worm is not possible. About the posterior 
part of the middle third of the parasite there is an irregular aggrega- 
tion of granular matter, which represents a viscus of some sort. With 
a high power one can further make out a delicate transverse striation 
in the musculocutaneous layer throughout the entire length of the 
animal. In stained specimens two V-shaped light-spots can be 
made out: one at a point about one-fifth of the entire length of the 
organism, backward from the head end ; the other, very much smaller, 
a short distance from the tail. The first Manson designates the 
"V" spot, the second the tail spot. In stained specimens these 
two spots are readily made out, as they do not take the color. When 
the movements of the animal have almost ceased, one can see on 
careful focussing that the head is constantly being covered and 
uncovered by a six-lipped or hooked and very delicate prepuce; 
and, moreover, one can sometimes see a short fang of extreme 
tenuity suddenly shot out from the uncovered extreme cephalic end, 
and as suddenly retracted. 

Technique. — The examination should be made late in the even- 
ing, after the patient has rested for a number of hours. Drops of 
blood are then mounted, wet, on slides and ringed with vaselin to 
prevent the specimen from drying. In such preparations the filarias 
keep alive for a week or longer. They should be searched for with 
a low power — an inch objective is very convenient for the purpose. 
Attention is directed to their presence by the commotion which they 
cause among the neighboring blood corpuscles. 

To prepare permanent mounts blood smears are best made on 
slides, which are then stained with eosinate of methylene blue in the 
usual manner. Working with the blood of infected animals, I have 
thus obtained very good results. The V and tail spots are very well 
brought out. To show anatomical details, however, staining with 
eosin and hematoxylin, after fixing the smears with alcohol, gives the 
best results; in this manner the sheath is very well shown, as also 
the structure of the musculocutaneous layer. 
13 



194 THE 'BLOOD 

The number of worms which may be found in a specimen is very 
variable. During the daytime they are rarely seen, and, if at all, 
only one or two specimens at most are found. As evening ap- 
proaches, however, commencing about 5 or 6 o'clock, the filarias 
enter the peripheral circulation in increasing numbers. At mid- 
night the maximum number is about reached, with from 300 to 600 
to the drop of blood. Later they gradually decrease, and by 8 or 
9 a.m. they have again disappeared. This periodicity, however, 
may be reversed if the patient is made to sleep during the daytime 
and remains awake at nights. During their absence from the 
peripheral circulation they may be found in the larger arteries and 
in the lungs. 

In non-active cases the number of filarias even at night is quite 
small. In one instance of this kind I found only the sheath of a 
single worm while examining perhaps fifty specimens. 

Infection occurs through the females of mosquitoes belonging to 
both the culex and anopheles family which have fed on the blood of 
filaria-infected individuals. The history of the parasite while in the 
body of the mosquito is in brief the following: After their arrival 
in the stomach the young worms shed the sheath and invade the 
thoracic muscles, where they increase in size (to 1.5 mm.), de- 
velop a mouth, an alimentary canal, and a trilobed tail. They 
then find their way into the abdomen, where, in suitably prepared 
sections, they may occasionally be seen in the tissues about the 
stomach, and even among the eggs in the posterior part of the 
abdomen. The majority now find their way to the base of the pro- 
boscis and under appropriate conditions out through the proboscis 
by a channel which they make for themselves. After introduction 
into the human body the organism finds its way into the lymphatics, 
where it attains sexual maturity; fecundation takes place and the new 
generation of filarias enter the blood current by way of the thoracic 
duct and the left subclavian vein. The development of the embryo 
form in the mosquito occupies from sixteen to twenty days. 

Whether or not infection can occur in any other way is not 
known. We could conceive that some of the worms are eliminated 
with the eggs of the mosquitoes, and that infection could then take 
place through contaminated drinking water. 

Filaria Perstans. — This species is of interest, as it was thought 
to be concerned in the causation of the so-called sleeping sickness of 
west tropical Africa. It has likewise been found in the Buck Indians 
of British Guiana, among whom the same sickness also occurs. 1 The 
organism observes no periodicity, but is present in the blood both 
during the daytime and at night. 

1 More recent observations tend to throw doubt on this relationship and rather 
suggest a connection between a species of trvpanosoma and sleeping sickness. 
(See p. 187.) 



ANIMAL PARASITES 



195 



The embryo worm is smaller than the Filaria nocturna; it meas- 
ures about 0.2 mm. in length by 0.004 mm. in breadth. It has 
no sheath, and its caudal end is truncated and abruptly rounded. 
There is no hooked cephalic prepuce. Its motion is progressive. 

The adult form measures 70 to 80 mm. in length. The tail in both 
sexes is incurvated and the chitinous covering at the extreme tip 
split, as it were, into two minute triangular appendages. They 
have been found in the connective tissue, at the root of the mesen- 
tery, behind the abdominal aorta, and beneath the pericardium. 

Literature. — Mosler u. Peiper, Spezielle Pathol, u. Therap., 1894, vol. vi, 
p. 219. P. Manson, Allbutt's S}'stem of Medicine, vol. ii. I. Guiteras, Med. 
News, April, 1886. F. P. Henry, ibid., 1896. E. Opie, Amer. Jour. Med. Sci., 1901, 
vol. cxxii, p. 251. P. Manson, Tropical Diseases, Cassell & Co., London, 1900. 




Fig. 55. — Male and female specimens of the human blood fluke {Bilharzia hcematobia). 
X 12. (After Looss.) 



Distomiasis (Bilharziasis) .—Bilharzia hsematobia (Cobbold): 
syn., Gynsecophorus (Diesing); Distomum haematobium (Bilharz); 
Schistosoma haematobium (Weinland); Distoma capense (Harley); 
Thecosoma (Maguin-Tandon). 

The Bilharzia hsematobia belongs to the class of trematode 
platodes. According to Bilharz, the greater portion of the Fellah 
and Coptic population of Egypt is infected. It is abundant in 
South Africa, and has also been observed in Mesopotamia, and 



196 THE BLOOD 

apparently in Arabia. In the United States a few isolated cases 
have been seen which were undoubtedly imported. From Europe 
no endemic cases have been reported. The parasite may give rise 
to diarrhea, hematuria, and ulceration of the mucous surfaces. 

The male is smaller but thicker than the female, measuring from 
12 to 15 mm. in length by 1 mm. in breadth. On its abdominal 
surface a deep groove is found with overlapping edges, which serves 
for 'the reception of the female (Fig. 55). It has an oval and a 
ventral sucker placed close together. 

The adult parasites are found in the blood of the portal vein, in 
its mesenteric and splenic branches, and in the vesical, uterine, and 





Fig. 56. — Bilharzia eg?s from the urine: Group a was drawn to scale with B. & L. % 
obj., and 1 in. ocular; group b represents their appearance with B. & L. % obj. 

hemorrhoidal veins; they have also been found in the vena cava 
and may possibly occur elsewhere in the circulation. The eggs 
are more often seen. They are oval bodies, measuring 0.16 mm. 
in length by 0.05 mm. in breadth, and are provided with a dis- 
tinct, spike-like projection which issues from one extremity or the 
side (Fig. 56). Infection usually takes place through unfiltered 
drinking water, but may also occur through the skin. " Through the 
portal system the parasite then invades the urogenital system, the 
anus, and rectum, and may also proliferate abundantly in the intes- 
tine, the liver, kidneys, etc. The diagnosis is usually made by 
examination of the urine, in which the ova will be found. 

Another variety of blood fluke has been described by J. Catto, 1 
Schistosoma Cattoi; it was found in a Chinese who had died of cholera. 

Literature. — Bilharz, Wien. med. Woch., 1856, vol. vi, p. 49. Meissner, 
Schmidt's Jahrbuch., 1882, vol. xx, p. 193. Rutimever, Verhandl. d. Cong. f. 
inn. Med., 1822, vol. xi, p. 144. 

Anguilluliasis* — In 1895 Teissier reported a case of intermittent 
fever in which numerous embryos of anguillula were found in the 

1 Brit, Med, Jour,, January 7, 1905. 



ANIMAL PARASITES 197 

blood. They disappeared after expulsion of the parasites from the 
intestinal tract, and at the same time the fever ceased. It is a question, 
however, whether Teissier's parasite was identical with the common 
form described by Bavay, Normand, Grassi, and others. Unlike 
the embryos developing from the eggs of both parasitic and free- 
living generations, Teissier's form did not present the characteristic 
double oesophageal enlargement, and he reports, moreover, that in 
the case of the adult male only one, instead of two, spicules was noted. 
This view is strengthened by the observation that after inoculation 
into frogs the worms developed in the intestinal canal and the lungs 
into giant forms, which may have been Ascaris nigrovenosa (syn., 
Rhabdonema nigrovenosum). 

Literature. — Teissier, Compt.-rend. del'Acad. des sci., 1895, vol. cxxi, p. 171. 
Arch, de med. exper. et d'anat. path., 1895, vol. vii, p. 675; ibid., 1896, vol. viii, 
p. 586. 



CHAPTER II. 
THE SECRETIONS OF THE MOUTH. 
SALIVA. 

Normal saliva is a mixture of the secretions derived from the 
submaxillary, sublingual, parotid, and mucous glands of the mouth. 
It is a colorless, inodorous, tasteless, somewhat stringy and frothy 
liquid, and serves the purpose of aiding in the acts of mastication, 
deglutition, and digestion. The quantity secreted in twenty-four 
hours amounts to about 1500 grams. 

General Characteristics. 

Normal saliva has a specific gravity of 1.002 to 1.009, correspond- 
ing to 4 to 10 grams of solids. The reaction is alkaline, the degree 
of alkalinity corresponding to from 0.006 to 0.048 per cent, of sodium 
hydrate. Normally an acid saliva is observed only in newly born 
infants and in sucklings. 

The reaction of the tongue and the mucous membrane lining the 
mouth is quite commonly acid early in the morning owing to the 
production of lactic acid by some of the bacteria which are constantly 
present in the mouth. This acid corrodes the enamel of the teeth, 
and may ultimately produce dental caries. 

Chemistry of the Saliva. 

In order to give an idea of the general composition of the saliva 
the following analyses are appended; the figures correspond to 1000 
parts by weight: 

Water 995.20 994.20 988.10 

Ptyalin 1 1.34 1.30 1.30 

Ep^helium} 162 220 260 

Fatty matter. ...... .. .. 0.50 

Sulphocyanides 0.06 0.04 0.09 

Alkaline chlorides 0.84 

Disodium phosphate .... 0.94 2.20 3.40 

Magnesium and calcium salts . . 0.04 

Alkaline carbonates .... traces. 

Nitrites traces. 

1 These figures are too high, as they refer to the total precipitate obtained with 
alcohol. 



SALIVA 199 

In order to demonstrate the presence of the sulphocyanides, it is 
usually only necessary to heat a few cubic centimeters of the pure 
saliva, faintly acidified with hydrochloric acid, with a dilute solu- 
tion of ferric chloride, when a red color will be seen to develop. 
If necessary, larger quantities, such as 100 c.c, are evaporated 
to a small volume; the test is then applied to the concentrated 
fluid. 

The test for nitrites is conducted in the following manner : About 
10 c.c. of saliva are treated with a few drops of Ilasvay's reagent 
and heated to a temperature of 80° C, when in the presence of nitrites 
a red color will develop. The reagent is prepared as follows: 0.5 
gram of sulphanilic acid in 150 c.c. of dilute acetic acid is treated 
with 0.1 gram of naphtylamin dissolved in 20 c.c. of boiling water. 
After standing for some time the supernatant fluid is poured off and 
the blue sediment dissolved in 150 c.c. of dilute acetic acid. The solu- 
tion is kept in a sealed bottle. 

Of organic matter, ptyalin, a little albumin mixed with mucin, and 
about 1 gram of urea pro liter are found. 

In neutral or slightly alkaline, but not in acid solutions ptyalin 
rapidly transforms boiled starch into dextrins and sugar at a tempera- 
ture of from 35° to 40° C. 

In order to test for ptyalin, sl few cubic centimeters of saliva are 
filtered and added to a solution of starch; the mixture is placed in 
the warm chamber for 5 to 10 minutes, when it is tested with cupric 
sulphate or iodine. At first starch gives a blue color with iodine; 
after digestion has proceeded farther a red or violet red is ob- 
tained, indicating the presence of erythrodextrin, while no change 
in color at all results when achroodextrin only is present. The 
maltose may be recognized by the fact that it turns the plane of 
polarization more strongly to the right than glucose; like glucose, it 
reduces Fehling's solution. 



Microscopic Examination of the Saliva. 

If normal saliva is allowed to stand, two layers will be seen to 
form, viz., an upper clear and a lower cloudy layer, which latter con- 
tains certain morphological elements. Among these, salivary cor- 
puscles, pavement epithelial cells, and microorganisms are found 
(Fig. 57). _ 

The salivary corpuscles resemble white corpuscles very closely, 
but differ in their greater size and coarser appearance. The epi- 
thelial cells are large, irregular, polygonal cells, provided with well- 
defined nuclei and nucleoli; they exhibit certain irregularities in 
size, according to their origin, and belong to the class of pavement 
or stratified epithelium. 



200 



THE SECRETIONS OF THE MOUTH 



Microorganisms. 1 — While schizomycetes and molds are only 
exceptionally found in the mouth under normal conditions, bacteria 
are always present in large numbers, and it is not surprising that all 
forms which are found in the air, food, and drink may here be encoun- 
tered. Some of these, such as the Leptothrix buccalis innominata, 
Bacillus buccalis maximus, Leptothrix buccalis maxima, Iodococcus 
vaginatus, Spirillum sputigenum, and Spirocheta dentium, are always 
present. Together with other bacteria, they have been found in 
carious teeth, in abscesses communicating with the mouth and 
pharynx, and in exudates on the mucous membranes of these parts. 
In all probability, however, they are non-pathogenic. To this <?lass 
also belongs the smegma bacillus, which has been encountered in the 
saliva, the coating of the tongue, and in the tartar of the teeth of per- 




Fig. 57. — Buccal secretion. (Eye-piece III, obj. Reichert, V 15 homogeneous immersion: 
Abbe's mirror, open condensers.) a, epithelial cells; b, salivary corpuscles; c, fat drops; d, 
leukocytes; e, Spirochaeta buccalis; f, comma bacillus of mouth; g, Leptothrix buccalis; h, i, 
k, various fungi, (v. Jaksch.) 

fectly healthy individuals. The Leuconostoc hominis also is a normal 
inhabitant of the oral cavity, but occurs in larger numbers in inflam- 
matory diseases (scarlatina, measles, and diphtheria). 2 

In this connection it is interesting to note that, in contradistinction 
to the bacteria which are only temporarily found in the mouth, the 
majority of those which are constantly present cannot be cultivated 
on artificial media. 

Important from a practical standpoint is the fact that a number 
of pathogenic microorganisms may be found under normal con- 
ditions. The Diplococcus pneumonias has thus been found in a 
virulent condition in from 15 to 20 per cent, of healthy individuals, 
and it is even claimed that in a non-virulent state it is constantly 
present in the mouth. Streptococci are likewise frequently observed, 
but usually possess but little virulence or none at all when obtained 
from the healthy mouth and tested upon animals. Pyogenic staphy- 

1 W. D. Miller, Die Mikroorganismen d. Mundhohle, 1892. 

2 Hlava, Folia hsematol., vol. i, p. 612. 



SALIVA 201 

lococci may also be found at times, but are less common than the 
streptococci. Most important is the occasional occurrence of the 
diphtheria bacillus in the mouths of individuals who have not been 
exposed to contagion. Welch 1 mentions that virulent organisms 
were found by Park and Beebe in the healthy throats of 8 out of 
330 persons in New York who gave no history of direct contact with 
cases of diphtheria; 2 of these 8 persons later developed the disease. 
Non-virulent bacilli were found in 24 individuals of the same series, 
and pseudodiphtheria bacilli in 27. 

Other pathogenic bacteria which may be found in normal mouths 
are the Micrococcus tetragenus, the Bacillus pneumoniae of Fried- 
lander, the Bacillus crassus sputigenus, and the Bacillus coli com- 
munis. 

Pathological Alterations. 

It has been mentioned that about 1500 grams of saliva are 
secreted in the twenty-four hours. This quantity is, however, sub- 
ject to great variation. An increase is thus frequently noted in 
pregnancy, in various neurotic conditions, in tabes, bulbar paralysis, 
in inflammatory diseases of the mouth, in dental caries, following 
the administration of pilocarpine, in poisoning with mercury, acids, 
and alkalies, etc. The quantity is diminished in all febrile diseases, 
in diabetes, and often in nephritis. The effect of psychic influences 
upon the secretion of saliva as well as of other glands is well known, 
an increase or decrease in the flow being produced under various 
conditions. 

In determining whether or not salivation actually exists, the physi- 
cian should not only be guided by the statements of the patient, 
but an actual estimation of the amount secreted within a definite 
period of time should be made. Nervous individuals not infrequently 
complain of "salivation," when a direct estimation will show that 
the amount is not only not increased, but actually diminished. 

An acid reaction has been noted in various diseases of the intestinal 
tract, in febrile diseases, and notably in diabetes. According to 
Strauss and Cohn, however, an alkaline reaction is the rule even under 
pathological conditions. 

Among the qualitative changes may be mentioned an increase in 
the amount of urea, which has been repeatedly observed in nephritis. 

Urea may be demonstrated as follows: The saliva is extracted 
with alcohol, the filtrate evaporated, and the residue dissolved in 
amyl alcohol. This is allowed to evaporate spontaneously, when 
crystals of urea will separate out, and may be further examined (see 
Urine). 

Bile-pigment and sugar have not been found in the saliva. 

1 Dennis' System of Surgery; Surgical Bacteriology. 



202 THE SECRETIONS OF THE MOUTH 



SPECIAL DISEASES OF THE MOUTH. 

Tuberculosis. — In cases of lupus and the so-called benign form 
of tuberculosis of the mouth it is rarely possible to demonstrate the 
presence of tubercle bacilli, even in scrapings taken from the base 
of the ulcers or in the diseased tissue itself, while in cases of ulcer- 
ative stomatitis associated with phthisis in its advanced stages they 
may be frequently found in large numbers. In some cases, however, 
their demonstration is by no means easy. In the saliva they are 
only exceptionally seen. 

Actinomycosis. — In cases of actinomycosis it is occasionally pos- 
sible to demonstrate the presence of the specific organism in or about 
carious teeth. More commonly, however, the patients are not seen 
until the primary symptoms of the disease have disappeared, when 
the typical kernels can no longer be found at the original points 
of entry or have become unrecognizable owing to calcification and 
retrogressive changes. 

Usually the disease has already progressed to the formation of a 
distinct tumor or abscess, and it may then be necessary to make an 
exploratory incision, and to examine the scrapings which are brought 
away. The number of kernels which may be found is at times very 
small, but a careful examination will probably always lead to their 
detection if the disease in question is actinomycosis. 

Catarrhal Stomatitis. — In this affection the quantity of saliva 
is increased. Microscopically an increased number of epithelial 
cells and many leukocytes are noted, their number depending upon 
the intensity of the morbid process. 

Ulcerative Stomatitis. — In this condition, following mercurial 
poisoning or scurvy, the same appearance is noted microscopically 
as in simple stomatitis. In addition there may be necrotic tissue, 
red blood corpuscles, and innumerable leukocytes. The reaction of 
the saliva is intensely alkaline, the color markedly brown, and its 
odor fetid. 

Gonorrheal Stomatitis. — The number of cases of gonorrheal 
stomatitis that have thus far been recorded is small. The disease, 
however, has received but little attention, and is probably more 
common than is generally supposed. In suspected cases the exudate 
which forms upon the gums, the tongue, and the palate should be 
examined for gonococci. 

Thrush. — Oiidium albicans (Fig. 58) is most commonly seen in 
children, but may also occur in adults, and especially in phthisical 
individuals, and sometimes lines the entire mouth. If in such cases 
a bit of the membrane is pulled off and examined microscopically, it 
will be found to consist of epithelial cells, leukocytes, and granular 
detritus, with a network of branching, band-like formations, which 



COATING OF THE TONSILS 



203 



present distinct segments. The contents of the segments are clear, 
and usually contain two highly refractive granules — the spores, one 




Fig. 58. — O'idium albicans, the vegetable parasite of thrush. (Reduced from Ch. Robin.) 

of which is situated at each pole. These segments diminish in size 
toward the end of each band, their contents at the same time becom- 
ing slightly granular. 

Tartar. — In a bit of tartar scraped from the teeth actively moving 
spirochetes are seen, as well as long, usually segmented bacilli, fre- 
quently forming bands which are colored bluish red by a solution of 
iodopotassic iodide. Leptothrix buccalis, shorter bacilli (which are 
not colored by this reagent) , micrococci, and a large number of leuko- 
cytes and epithelial cells which have undergone fatty degeneration, 
are also found. Infusoria have been found by Sternberg, P. Cohn- 
heim, v. Leyden, and others. 



COATING OF THE TONGUE. 

A brown coating of the tongue is often observed in severe infectious 
diseases, and consists of remnants of food and incrustated blood. 
Microscopically, in addition to a large number of epithelial cells, 
enormous numbers of microorganisms and a large number of dark, 
cell-like structures, probably derived from desquamated epithelial 
cells, are found. The white coating of the tongue contains epithelial 
cells, many microorganisms, and a few salivary corpuscles. 



COATING OF THE TONSILS. 



Pharyngomycosis Leptothrica. — In the pyoid masses derived from 
the crypts of the tonsils in cases of follicular tonsillitis, and also in 
persons who have had frequent attacks of tonsillitis, large numbers 
of lymphocytes of all sizes are seen, besides epithelial cells and long, 



204 



THE SECRETIONS OF THE MO UTE 



segmented fungi — the Leptothrix buccalis (Fig. 59) — which are 
colored bluish red by a solution of iodopotassic iodide. Ordinary 
polynuclear neutrophiles are only present in small numbers. At 
times patches composed of these fungi extend over a considerable 
area of the tonsils, so that it may be doubtful whether or not the 
disease is a beginning diphtheria. 

More extensive invasions have been described by Dubler, who noted 
a leptothrix mycosis involving the pharynx, esophagus, and larynx; 
and by Baginsky in the case of the pharynx, trachea, and nose. 

Literature. — Frankel, Berlin, klin. Woch., 1873, p. 94. Miller, Die Mikro- 
organismen der Mundhohle, 1889, Leipzig. Stern, Munch, med. Woch., 1893 
p. 381. Hering, Zeit. f. klin. Med., 1884, p. 358. Dubler, Virchow's Arch.^ 
1891, vol. cxxvi, p. 454. Baginsky, cit. by Hering (vide supra). 




Fig. 59. — Leptothrix buccalis. (v. Jaksch.) 

Tonsillitis. — In tonsillitis a large number of bacteria have been 
isolated fiom the pseudomembranous deposits. Among the more 
important which are supposed to bear a causative relation to the dis- 
ease may be mentioned the various streptococci, staphylococci, less 
commonly the pneumococcus, the Micrococcus catarrhalis, the Bacillus 
coli communis, the bacillus of Friedlander, the Bacillus septicaemia? 
sputi, and in a few isolated instances the Micrococcus tetragenus. 
In many cases in which tonsillar deposits are clinically regarded as 
diphtheritic culture reveals only an abundance of the thrush fungus. 

Meyer, 1 in v. Leyden's clinic, succeeded in cultivating a diplo- 
streptococcus from the tonsils in five cases of acute rheumatism with 
angina, and reports that bouillon cultures of the organism produced 
characteristic polyarticular arthritis in rabbits. The same organism 
apparently was also obtained by Allaria 2 in Bozzolo's clinic, and it 
is interesting to note that his cases resulted from manifest contagion. 



1 Deutsch. med. Woch., 1901, vol. xxvii, p. 81. 

2 Revista critica di clinica Medica, 1901, vol. ii, p. 805. 



COATING OF THE TONSILS 205 

Vincent's Angina. — In cases of Vincent's angina (ulceromembranous 
angina and stomatitis) smears from the exudate will be seen to contain 
innumerable organisms which are essentially of two types, viz., spirilla 
and long, fusiform bacilli (Fig. 60). Occasionally, though exception- 
ally, the bacilli only may be found. The spirilla usually present three 
or four convolutions and are generally actively motile. They measure 
from 36 to 40 fi in length by 0.5 (jl in breadth. The bacilli measure 
from 6 to 12 fi in length and are somewhat stouter in the middle 
than at the ends. They may occur in twos, joined end to end, and 
usually scattered uniformly throughout the preparation. They are 
non-motile. Spirilla and bacilli are readily stained with a dilute 
solution of carbol fuchsin (1 to 20), which should be filtered before 
use. Loffler's blue and gentian-aniline water may likewise be used. 

The bacilli are obligate anaerobes ; the spirilla may be obtained 
together with the bacilli in mixed cultures. 

Of late the opinion has been expressed that the spirilla and bacilli 
may represent stages in the life history of a trypanosome. 

Both organisms have occasionally been found associated with 
diphtheria bacilli. 

The disease seems to be more common than was first thought. 
The earlier cases were reported by Vincent, Bernheim, Conrad, and 
others. In the United States the disease has been described by 
Mayer, Fisher, Crandall, Weaver and TunniclifT, Berkeley, and others. 

Literature. — J. W. Byers, Lancet and Brit. Med. Jo-urn., January 9, 1904. 
Weaver and Tunnicliff, Journ. of Infect. Dis., 1905 vol. ii, p. 446. Berkeley, 
Med. News, 1905, vol. xxxvi, p. 976. Wright, 1904, July 4, p. 73. 

Diphtheria. — Recognizing the great importance of an early diagnosis 
in cases of diphtheria, an examination for Loffler's bacillus has become 
just as important today as that for the bacillus of tuberculosis. 

By means of a stout platinum loop, a pair of forceps, or a cotton 
swab, a piece of membrane is scraped from the tonsils, the soft palate, 
or the pharynx. From this cultures are prepared as described below ; 
at the same time smears are made on slides and fixed, when air dry, by 
being passed several times through the flame of a Bunsen burner. 
They are then stained for five to ten minutes in Loffler's alkaline 
solution of methylene blue, which consists of 30 c.c. of a concentrated 
alcoholic solution of methylene blue in 100 c.c. of an aqueous solution 
of potassium hydrate (1 to 10,000). They are then rinsed in water, 
dried and examined with a yy oil-immersion lens. 

A rapid method of staining, and one which also gives satisfactory 
results, is suggested by Neisser. The organism is grown on ox-blood 
serum and examined after nine to twenty-four hours. The air-dried 
smears are placed for one to three seconds in a solution composed of 
20 c.c. of an alcoholic solution of methylene blue (1 to 20 c.c. of 90 
per cent, alcohol), 950 c.c. of distilled water, and 30 c.c. of glacial 



206 THE SECRETIONS OF THE MOUTH 

acetic acid. They are then washed in water, stained for three to 
five seconds in a 0.2 per cent, hot and filtered aqueous solution of 
vesuvin, again washed off, dried in the air, and mounted in balsam. 
The bacilli are brown and have in their interior 2 to 4 blue granules 
which are usually located near the poles. 

The following method also may be employed, as suggested by 
Schauffler. The staining reagent has the following composition : 

Filtered solution of Loffler's methylene blue 10.0 c.c. 

Filtered solution of pyronin (0.5 gram to 10 c.c. of water) . 1.5 c.c. 
Acid alcohol (3 c.c. of 25 per cent hydrochloric acid to 97 c.c. 

of absolute alcohol) 0.5 c.c. 

Cover-glass specimens are stained for one minute; they are then 
washed in running water and mounted in balsam as usual. The 
bacilli are stained blue, the pole bodies a bright ruby red. 

Pseudodiphtheritic bacilli are said to take only the blue stain with 
this method. 

The organism grows best on Lofflers blood serum; upon this it 
develops so much more rapidly than other organisms which are usually 
present in the secretions of the mouth and throat, that, after six to 
eight hours' incubation at 34° to 35° C, it often forms the only colonies 
that attract attention. Smears are then made and stained according 
to Neisser's or Loffler's method. 

In the absence of blood serum, bouillon, nutrient gelatin, nutrient 
agar, glycerin agar, and potato may be employed. Coagulated 
egg albumen, as pointed out by Booker, and milk are also good media. 
But it is to be noted that the "typical" staining effect with Neisser's 
method is commonly only obtained if the organism has been grown 
on ox-blood serum, and if the growth is not older than twenty-four 
hours. 

According to Knapp the true bacilli, in contradistinction to the 
pseud odiphtheria bacilli, will ferment dextrose and maltose. The 
Bacillus xerosis will do the same. In contradistinction to the 
diphtheria organism the Bacillus xerosis will ferment cane sugar; 
the former, in contradistinction to the xerosis, will ferment dextrin. 
The fermentation tests must be made with the litmus serum-water 
media of His. 1 Results after twenty-four hours' growth at 37° C. : 
Pseudodiphtheria — none of the sugars fermented; media remain 
blue. Diphtheria — dextrose, mannite, maltose, and dextrin fer- 
mented; media red and coagulated. Saccharose not fermented. 
Xerosis bacillus — dextrose, mannite, maltose, and saccharose fer- 
mented with acid production; media red and coagulated. Dextrin 
not fermented. The Bacillus xerosis, moreover, forms a very thin 
scum or pellicle on the surface of the media which is absent with 
the other bacteria. 

1 Journ. of Med. Res., vol. xii, p. 475-478. See also Appendix: Media. 



COATING OF THE TONSILS 



207 



The colonies are large, round, elevated, and grayish white in 
color, with a centre that is more opaque than the slightly irregular 
periphery. The surface of the colony is at first moist, but after a 
day or two it assumes a dry appearance. 







* 


< 


>> 


\ 


~~~~~~ • r 


/ 1 


W 


•'' "> J 


. / 


>i& 


! 


*• 


;■ ' / ■ 


1 V 

y 




1 7 








V 




Fig. 60. 



Fig. 61. 





Fig. 62. Fig. 63. 1 

Fig. 60. — Spirilla and fusiform bacilli of Vincent's angina. 

Fig. 61. — Characteristic forms of diphtheria bacilli from blood-serum cultures, showing 
clubbed ends and irregular stain. X 1100 diameters. (Park.) 

Fig. 62. — B. diphtheria*. Forty-eight hours' agar culture. Thick, medium-clubbed rods and 
moderate number of segments. One year on artificial culture media. X 1410 diameters. 
(Park.) 

Fig. 63. — Colonies of diphtheria bacilli. X 200 diameters. (Park.) 



The bacillus (Figs. 61, 62, and 63) is non-motile and varies in size and 
shape, its average length being from 2.5 to 3 //., its breadth from 0.5 to 
0.8 p.. Its morphological characteristics are so peculiar as to render 
its identification upon cover-slip preparations and in sections of the 
diphtheritic membrane an easy matter in most cases. 



208 THE SECRETIONS OF THE MOUTH 

Sometimes the organism appears as a straight or slightly curved 
rod; but especially characteristic are irregular and often bizarre 
forms, such as rods with one or both ends terminating in little bulbs 
and rods apparently broken at intervals, in which short, well- 
defined, round, oval, or straight segments can be made out. Very 
commonly two organisms lie together forming an obtuse angle, or 
numbers of them may be observed lying side by side. 

Some forms stain uniformly, others in an irregular manner; the 
most typical appearance is that of little granules near the poles of 
the bacillus, which stain blue with Neisser's method, while the body 
of the organism is colored brown. 

Streptococci are also seen as a rule, and it may be said that the 
gravity of a case is directly proportionate to the number of strep- 
tococci present. 

It is important to note that diphtheria bacilli may still be found 
in the throat for weeks after all clinical symptoms have disappeared. 
Patients should hence be isolated until a bacteriological examination 
has demonstrated the absence of the organism. 

Literature. — S. Flexner, "The Bacteriology and Pathology of Diphtheria,'' 
Johns Hopkins Hosp. Bull., 1895, p. 39. W. H. Welch, Amer. Jour. Med. Sci., 
1894. Heubner, Schmidt's Jahrbucher d. gesammten Med., 1892, vol. ccxxxvi, 
p. 270. Klebs, Arch. f. exper. Path., 1875, vol. iv, p. 207. Loffler, Centralbl. 
f. Bakt. u. Parasit., 1887, vol. ii, p. 105: and 1890, vol. vii, p. 528. C. Frankel, 
"Die Unterscheidung d. echten u. d. falschen Diphtheriebacillen," Berlin, klin. 
Woch., 1897, p. 1087. W. G. Schauffler, Med. Record, December 6, 1902. 

Scarlatina. — According to Baginsky, streptococci are practically 
constantly found in the pharyngeal secretion. 

Literature. — A. Baginsky, Deutsch. med. Woch., October 23, 1902. 

Glandular Fever. — According to Neumann and Comby, glandular 
fever generally depends upon infection with a streptococcus. In the 
case reported by Lande and Froin and by Hirtz 1 bacteriological 
examination of the throat at the height of the febrile stage revealed 
the presence of the pneumococcus in a virulent condition. 

1 Lande et Froin, Rev. mensuelle des Mai. de l'Enfance, 1901, p. 78. 



CHAPTER III. 

THE GASTRIC JUICE AND GASTRIC CONTENTS. 

THE SECRETION OF GASTRIC JUICE. 

The gastric juice is the result of the glandular activity of the 
stomach, and is the only secretion of the digestive tract which pre- 
sents an acid reaction. 

As is well known, the mucous membrane of the stomach is cov- 
ered throughout its entire extent by a single layer of cylindrical 
epithelium, which dips down in places to line the orifices and larger 
ducts of the numerous tubular glands with which it is beset. Of 
these, two kinds are described, viz., the fundus and pyloric glands, 
so named from the location in which they are principally found. 
In the secretory portion of a fundus gland two sets of cells can be 
distinguished. The one kind is small, granular, and polyhedral or 
columnar, bordering upon the narrow lumen of the tube; these are 
termed the chief or principal cells (Heidenhain), but are also known 
as the central or adelomorphous cells. They stain with aniline dyes 
to only a slight extent. The others, known as parietal, adelomor- 
phous, 01 oxyntic cells, are variously situated between the adelomor- 
phous cells and the membrana propria; they are most numerous 
in the necks of the glands. They are larger than the chief cells, oval 
or angular and finely granular in structure; they possess a strong 
affinity for the aniline dyes. The pyloric glands, which are found 
only in the region of the pylorus, on the other hand, are character- 
ized by the greater length of their ducts, which are also lined by 
the cylindrical epithelium of the mucous membrane proper. The 
secretory portion of these glands is represented by a single layer of 
short and finely granular, columnar cells, which closely resemble the 
chief cells of the fundus glands. In addition to these, a few isolated 
cells, the cells of Nussbaum, are found, which in structure and in 
their behavior to aniline dyes resemble the parietal cells. 

Upon chemical examination the gastric juice is found to consist 
essentially of water, free hydrochloric acid, pepsin, rennet (a milk- 
curdling ferment), lipase, mucus, and certain mineral salts. 

Of these constituents hydrochloric acid is secreted by the parietal 

cells, pepsin, the milk-curdling ferment, and lipase by the chief 

cells of the fundus and the pyloric gland's, while the mucus is the 

product of the cylindrical goblet-cells lining the stomach and the 

14 



210 THE GASTRIC JUICE AND GASTRIC CONTENTS 

wider portions of its glandular ducts. It should be borne in mind 
that the ferments do not exist in the cells as such, but as zymogens, 
which are transformed into the ferments through the activity of the 
free hydrochloric acid. According to modern investigations, more- 
over, the zymogens only are secreted by the cells. 

Until recently it was supposed that the gastric juice is secreted only 
upon appropriate stimulation of the nervous mechanism of the stom- 
ach, either directly or indirectly, and that the stomach in its quiescent 
state — i. e.y when not digesting — is empty. The researches of 
Schreiber and Martius, however, have rendered the correctness of 
this view doubtful, as they were able to obtain quantities of gastric 
juice, varying from 1 to 60 c.c, from the non-digesting stomach of 
every normal person examined. 

Test Meals. — As the amount of hydrochloric acid which is 
secreted varies with the amount and the character of the food 
ingested, it has been found useful for purposes of comparison to 
make analyses after the administration of test meals of constant 
composition. The most important test meals are the following: 

The Test Breakfast of Ewald and Boas. — This consists of 35 
grams of wheat bread and 400 c.c. of water or weak tea, without 
sugar. It is best to give this meal to the patient early in the morning, 
when the stomach is empty — i. e., as a breakfast, and in cases of 
dilatation or of marked atony, after previous lavage. The gastric 
contents are obtained one hour later. 

The Test Breakfast of Boas. — This consists of a plateful of 
oatmeal soup, prepared by boiling down to 500 c.c. one liter of water 
to which one tablespoonful of rolled oats has been added. A little 
salt may be used if desired, but nothing more. The contents of the 
stomach are obtained one hour later. This test meal was devised 
by Boas in order to guard against the introduction from without of 
lactic acid, which is present in all kinds of bread. The meal is em- 
ployed in cases of suspected cancer of the stomach in which a quanti- 
tative estimation of lactic acid is to be made, the stomach being washed 
out completely the night before. 

The Test Dinner of Riegel. — This consists of a plate of soup 
(400 c.c), a beefsteak (150 to 200 grams), and 150 grams of mashed 
potatoes. The contents of the stomach are obtained after four 
hours. The disadvantage of this method lies in the fact that the 
lumen of the stomach tube is frequently occluded by pieces of undi- 
gested meat, a source of annoyance which may be guarded against 
by using finely chopped meat. Moreover, a positive lactic acid reac- 
tion (referable to sarcolactic acid) is obtained in a large number of 
cases, and entirely irrespective of the amount of hydrochloric acid 
present. 

The Double Test Meal of Salzer. — For breakfast the patient receives 
30 grams of lean, cold roast, hashed or cut into strips sufficiently 



THE SECRETION OF GASTRIC JUICE 



211 



small not to obstruct the stomach tube; 250 c.c. of milk; 60 grams of 
rice, and 1 soft-boiled egg. Exactly four hours later the second meal 
is taken, consisting of 35 to 70 grams of stale wheat bread and 300 
to 400 c.c. of water. The gastric contents are withdrawn one hour 
later. In this manner the gastric juice is not only obtained at the 
height of digestion, but an idea may at the same time be formed of 
the motor power of the stomach. Under normal conditions the organ 
should contain no remnants of the first meal at the time of examination. 

The Stomach Tube. — The stomach tubes in general use are 
essentially large Nelaton catheters. They should measure from 72 
to 75 cm. in length, and be provided with three fenestra, of which one 
is placed at the end of the tube and two laterally, as near the end as 
possible. For the purpose of washing out the stomach the tube is 
connected with a glass funnel. 

It is important that the tubes should be thoroughly cleansed in 
hot water as soon after use as possible. The advice of Boas, more- 
over, to have special marked tubes for tuber- 
culous, syphilitic, and carcinomatous patients 
should be borne in mind. - Patients in whom 
lavage is to be practised for any length of 
time should provide their own instruments. 

Contra-indications to the Use of the Tube. — 
Of direct contra-indications to the use of the 
tube there should be mentioned the existence 
of the various forms of valvular disease when 
in a state of imperfect compensation, angina 
pectoris, arteriosclerosis of high degree, aneu- 
rysm of the large arteries, recent hemorrhages 
from whatever cause, marked emphysema 
with intense bronchitis, acute febrile dis- 
eases, etc. 

Introduction of the Tube. — The technique 
of the introduction of the tube should be 
as simple as possible; the exhibition of 
complicated bottle arrangements for the 
purpose of obtaining the gastric juice only 
adds to the excitement of a nervous patient, 
and should be avoided. The patient's cloth- 
ing and floor of the room should be pro- 
tected from being soiled by material that 
may be vomited along the sides of the tube, 
the dribbling of saliva, etc. For this purpose, 
with pouch may be advantageously employed. 

Cocainization of the pharynx is not necessary, but may be resorted 
to in hyperesthetic individuals, a 10 per cent, solution being employed. 

1 Manufactured by G. Tiemann & Co., New York. 




Fig. 64. — Boas' bulbed tube. 



Turk's rubber bib 3 



212 



THE GASTRIC JUICE AND GASTRIC CONTENTS 



The tube, held like a pen, is passed to the posterior wall of the 
pharynx, the patient bending his head forward, and not backward, 
as is usually advised. The patient is then told to swallow. The 
tube is pushed until resistance is felt when it meets with the floor of 
the stomach. At the least sign of cyanosis or of marked pallor the 
tube should be withdrawn at once, and the patient observed for a 
day or two before a second attempt is made. 

If the gastric juice does not flow at once, the patient is instructed 
to bear down with his abdominal muscles, and, if this is insufficient, 
to cough a little. Repeated attempts of this kind will usually bring 
about the desired result, unless the tube has not been introduced far 
enough or too far; in the latter case it will double upon itself, so 
that its end rises above the level of the liquid. Pressing upon the 
abdomen with the hands is of no effect (Method of Expression). 

Aspiration must at times be employed. For this purpose Boas' 
bulbed tube (Fig. 64) is convenient. The manner in which it is used 
is the following: The proximal end of the tube, after having been 




Fig. 65. — Arrangement of a bottle for aspiration of the gastric contents. 



introduced into the stomach, is compressed and the bulb squeezed, 
when the distal end is clamped and the bulb allowed to expand. A 
partial vacuum is thus produced, which usually has the desired effect. 
In the absence of such an instrument the stomach tube may be con- 
nected with a bottle, in which a partial vacuum has been established 
by aspiration (Fig. 65). Unless the patient is accustomed to the intro- 
duction of the tube, however, these more complicated procedures 
should be avoided as much as possible (Method of Aspiration). 

In order to wash out the stomach, the funnel is filled with lukewarm 
water or any desired medicated solution, elevated above the head of 
the patient, and the water allowed to flow. From 500 to 1000 c.c. may 
be introduced at one time. By depressing and inverting the funnel 
over a suitable vessel before all water has left the funnel a siphon 



GENERAL CHARACTERISTICS OF THE GASTRIC JUICE 213 

arrangement is established and the stomach emptied. It is well to 
measure the returning water as well as the amount introduced. Should 
the flow diminish or cease before all the water has been removed, the 
end of the tube probably stands above the level of the liquid, and the 
flow can be started again by pushing the tube on farther or by with- 
drawing it a little, as the case may be. 

Washing out the stomach soon after the ingestion of a full meal is 
always very tedious and annoying, if not an impossible procedure, 
as the fenestra readily become obstructed. Should this occur, the 
funnel, filled with water, is elevated as high as possible, with a view 
to overcome the obstruction by hydrostatic pressure ; or, if this proves 
insufficient, the funnel is detached and the obstruction is lodged by 
means of air, for which purpose a Politzer bag or the bulb of a Boas 
tube is very convenient. 



GENERAL CHARACTERISTICS OF THE GASTRIC JUICE. 

Pure gastric juice is an almost clear, faintly yellowish fluid, of a 
sour taste and a peculiar characteristic odor. Its specific gravity 
varies between 1.002 and 1.003, corresponding to about 0.5 per cent, 
of solids. Its reaction, owing to the presence of hydrochloric acid, 
is acid. 

Amount. — Very little is known of the total quantity of gastric juice 
that is secreted in the twenty-four hours. The figure given by Beau- 
mont, 1 viz., 180 grams pro die, based upon observations made upon 
the often-quoted Canadian hunter, Alexis St. Martin, is undoubtedly 
too low. The amount given by Bidder and Schmidt, 2 viz., that corre- 
sponding to about one-tenth of the body weight, is probably more 
nearly correct. 3 It may be stated a priori that the quantity secreted 
varies within wide limits, being influenced by numerous factors, 
notably by the degree of the appetite and the amount and character 
of the food taken, especially that of the proteids. The age and sex 
of the individual, the time of day (notably in its relation to the 
ingestion of food), the emotions, etc., all influence the glandular 
activity of the stomach. 4 

From the non-digesting organ from 1 to 60 c.c. of gastric juice 
may be obtained at one time. The amount which can be procured 
during the process of digestion, on the other hand, varies with the 
amount of liquid ingested, the time of expression, the size and motor 

1 Experiments and Observations on the Gastric Juice, Boston, 1834. 

2 Verdauungssafte u. d. Stoffwechsel, 1852. 

3 Griinewald's figure — i. e., 1580 grams — I likewise regard as too low. 
According to my experience, the daily secretion appears to vary between 2000 
and 3000 c.c. 

4 See C. E. Simon, Physiological Chemistry, third edition, 1907, Lea Bros. 
& Co. 



214 THE GASTRIC JUICE AND GASTRIC CONTENTS 

power of the stomach, and the degree of transudation; the process 
of resorption probably does not play any part, as it has been ascertained 
that very little water, if any, is absorbed in the stomach. 

As a rule from 20 to 50 c.c. of filtrate can normally be obtained 
one hour after the ingestion of Ewald's test breakfast. 

Abnormally large quantities of gastric juice are practically found 
only in cases of so-called hypersecretion, the "Magensaftfluss" of 
the Germans, which may occur periodically or continuously. For- 
merly the presence of appreciable quantities of gastric juice in the 
non-digesting organ was regarded as conclusive evidence of the 
existence of this condition, but in the light of Schreiber's researches 
this position can no longer be maintained. The diagnosis should, 
hence only be made when in conjunction with the clinical symptoms 
of hypersecretion from 100 to 1000 c.c. of pure gastric juice can be 
obtained from the non-digesting organ. To this end, the stomach 
should be emptied completely by the tube before retiring, and an 
examination made on the following morning, no foods or liquids 
being allowed in the mean time. 

In various pathological conditions abnormally large quantities of 
liquid may be obtained, which cannot be regarded as gastric juice, how- 
ever. Attention will be drawn to these conditions at another place. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 

The Acidity of the Gastric Juice is Referable to the Presence 
of Free Hydrochloric Acid. — It has been conclusively demonstrated 
by Schmidt that the acidity of the gastric juice is due to the presence 
of free hydrochloric acid and to this only. After accurately deter- 
mining the amount of chlorine and all basic substances present, it 
was found that after the latter had been saturated a quantity of 
hydrochloric acid still remained, which in the dog varied between 
0.25 and 0.42 per cent., with an average of 0.33 per cent. The 
amount of free acid was also determined by titration and the same 
results reached as by gravimetric analysis. 

While it can thus be regarded as an established fact that hydrochlo- 
ric acid only is found in the gastric juice, such as it is secreted, there 
can be no doubt that traces of lactic acid may be found in the stomach 
contents during the process of digestion. These traces, however, have 
been introduced from without. 

The time at which hydrochloric acid will appear in the free state 
depends ceteris paribus upon the quantity of albumins ingested. 
With Ewald's test breakfast it appears after thirty-five minutes and 
reaches its maximum between fifty and sixty minutes after eating. 
With Riegel's meal the time is longer; it appears after one hundred 
and twenty to one hundred and fifty minutes in the free state and 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 215 

reaches its maximum after one hundred and eighty to two hundred 
and ten minutes. 

Under pathological conditions the amount of free hydrochloric 
acid, as will be shown, may undergo great variations, diminishing on 
the one hand to zero, and increasing on the other to 0.5 per cent., or 
even more. In other cases lactic acid and other organic acids may 
appear in notable amounts. 

Method of Determining the Total Acidity of the Gastric Con- 
tents. — To this end a known quantity of gastric juice is titrated with 
a one-tenth normal solution of sodium hydrate, using phenolphthalein 
as an indicator, when the number of cubic centimeters of the one- 
tenth normal solution employed, multiplied by the equivalent of 1 c.c. 




Total acidity. 



Free HC1. 



Combined acids. 



# 

Last lavage. Stomach empty 



Last expression (with chemical 
investigation). 



90 95 Minutes. 



Fig. 66. — Course of the acidity of the gastric juice after a test meal of 300 grams of 
tea and 50 grams of bread. (Schiile.) 

of this solution in terms of hydrochloric acid, will indicate the amount 
of acid present, from which the percentage acidity is readily cal- 
culated. 

Method. — 5 or 10 c.c. of filtered gastric juice are titrated with 
the one-tenth normal solution of sodium hydrate, using 2 or 3 
drops of a 1 per cent, alcoholic solution of phenolphthalein as an 
indicator until a permanent rose color appears. The number of 
cubic centimeters of the one-tenth normal solution employed multi- 
plied by 0.00365 will indicate the acidity of the 5 or 10 c.c. of gastric 
juice in terms of HC1, from which the percentage acidity is calcu- 
lated. 

Example. — 10 c.c. of gastric juice required the addition of 6.5 
c.c. of the one-tenth normal solution; 6.5 X 0.00365 (i. e., 0.0237) 
would hence indicate the acidity of the 10 c.c. of gastric juice in 
terms of HC1, and 0.0237 X 10 = 0.237, the percentage acidity. 

Or the result may be expressed in terms of the number of c.c. of 



216 THE GASTRIC JUICE AND GASTRIC CONTENTS 

the T \ solution which would be necessary to neutralize 100 c.c. of 
stomach contents. In the example the total acidity would thus be 
6.5 X 10 = 65. This method of indicating results is indeed the 
usual. 

Under normal conditions figures varying from 40 to 60 are usually 
obtained one hour after the ingestion of Ewald's test breakfast, 
while in pathological conditions greater variations are observed. 
In acute and chronic inflammatory conditions of the stomach, as 
well as in some of the neuroses, the acidity of the gastric contents 
is below normal. Higher figures are met with in some cases of ulcer 
and in some cases of dilatation, but are especially common in neurotic 
conditions; a degree of acidity corresponding to 90 or even more 
is then not infrequently observed. Increased acidity, usually asso- 
ciated with hypersecretion of gastric juice, is met with in the so- 
called hyper seer etio acida et continua of Reichmann. 

Preparation of decinormal alkali solution. — A normal solution of 
sodium hydrate is one containing the equ valent of its molecular 
weight in grams — i. e., 40 grams- — in 1000 c.c. of distilled water; a 
decinormal solution will, therefore, contain 4 grams in the same 
volume of water. This quantity is dissolved in about 900 c.c. and 
the solution brought to the proper strength by titrating it with a 
solution of oxalic acid of known strength. 

From the equation 

2NaOH + C 2 H 2 4 = C 2 Na 2 4 + 2H 2 0, 

it is seen that 2 molecules of NaOH (molecular weight 40) com- 
bine with 1 molecule of C 2 H 2 4 + 2H 2 (molecular weight 126), 
or 4 parts by weight of the former with 6.3 of the latter. 6.3 grams 
of chemically pure, crystalline oxalic acid (which is stable and non- 
deliquescent) are dissolved in 1000 c.c. of distilled water; this makes 
a T \ normal solution of the acid. Were the alkali solution of the 
proper strength it should take just 10 c.c. to neutralize 10 c.c. of 
the acid. But as the alkali solution cannot be made up accurately 
from the start (owing to inconstant weight from deliquescence), and as 
it has been purposely made too strong, less than 10 c.c. will be re- 
quired, e. g., 8 c.c. It is then ascertained how many such portions of 
alkali solution there are left, and then a corresponding amount of 
water is added, i. e., an amount representing the deficit found as 
compared with the acid solution. 

In the present example, for instance, we started with 900 c.c. of 
the uncorrected alkali solution, of which 8 c.c. were used in the test 
titration. There are remaining then 892 c.c. For every 8 c.c. in 
this bulk, viz., 111.5 portions, 2 c.c. of distilled water must be added; 
hence 111.5 X 2 = 223 c.c. A second titration is made to ensure the 
correctness of the result. 

Since 1000 c.c. of the one-tenth normal solution containing 4 grams 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 217 

of NaOH are equivalent to 3.65 grams of HO, as is seen from the 
equation 

NaOH +HC1 - NaCl +H 2 
40 36.5 

1000 c.c. of the T : o normal solution represent 3.65 grams of HC1 

100 " " " " " " 0.365 gram " " 

10 " " " " " " 0.0365 " " " 

1 " " " " " represents 0.00365 " " " 

It has been pointed out that the reaction of normal gastric juice 
is always acid, owing to the presence of free hydrochloric acid, and 
the same may be said to hold good for the gastric contents in general 
obtained from normal individuals. Pathologically an acid reaction 
is also the rule, as in those cases in which hydrochloric acid is absent 
fatty acids and lactic acid usually make their appearance. It is, 
therefore, not surprising that an alkaline, neutral, or amphoteric 
reaction is but rarely, or at least not commonly, observed in the 
gastric contents artificially obtained, and practically seen only in 
the so-called mucous form of chronic gastritis, or in those rare cases 
of anadeny, in which a complete destruction of the gastric glands 
has taken place. In vomited material, on the other hand, such 
observations are common, owing to the presence of large amounts 
of saliva. The vomited material in cases of so-called vomitus 
matutinus, which is usually referable to a chronic catarrhal condition 
of the pharynx, generally presents an alkaline reaction, owing to 
the fact that the fluid brought up is largely unchanged saliva. 

The Amount of Free Hydrochloric Acid.— Pure gastric juice, 
according to Ewald, 1 Szabo, 2 and Boas, 3 contains from 2 to 3 pro 
mille of free hydrochloric acid. 

In the digesting organ such amounts are met with only at the 
height of digestion, and after all basic affinities have been saturated. 
The time at which free hydrochloric acid can be demonstrated in 
the gastric contents after the ingestion of a meal will, hence, vary 
with the character of the food and its amount. When but little work 
is to be accomplished free hydrochloric acid is found much sooner 
than otherwise. After Ewald's test breakfast, it appears in thirty- 
five minutes; the point of maximum acidity is reached after from 
fifty to sixty minutes, and corresponds to 1.7 pro mille. Following 
RiegePs meal, on the other hand, the free acid appears after one hun- 
dred and thirty-five minutes, and reaches its highest point (corre- 
sponding to 2.7 pro mille) in from one hundred and eighty to two 
hundred and ten minutes. 

Clinically it is necessary to distinguish between euchlorhydria, or 
the secretion of a normal amount of free hydrochloric acid (0.1 to 

1 Loc. cit. 

2 Zeit. f. physiol. Chem., 1877, vol. i, p. 155. 

3 Loc. cit. See also A. Schule, Zeit. f. klin. Med., 1896, vols, xxvili and 
xxix. 



218 THE GASTRIC JUICE AND GASTRIC CONTENTS 

0.2 per cent.), hypochlorhydria, or the secretion of a deficient amount 
(less than 0.1 per cent.), hyperchlorhydria, in which more than 0.2 
per cent, is found, and anachlorhydria, in which no hydrochloric acid 
at all is secreted. 

Euchlorhydria. — Euchlorhydria, when associated with clinical symp- 
toms pointing to gastric derangement, is most commonly observed 
in gastric neuroses. A chronic gastritis can always be excluded 
in the presence of a normal amount of free acid. It may be 
associated with a certain degree of atony. It was formerly thought 
that a normal amount of acid would preclude the diagnosis of ulcer, 
but it is known that this association is quite possible. The same is 
seen in pyloric stenosis due to a healed ulcer. 

Hypochlorhydria. — Hypochlorhydria is associated with all those 
diseases in which the secretory elements have been more or less 
damaged, as the result of general disease (anemia, chronic heart 
and renal lesions, phthisis, chronic icterus, many febrile diseases), 
or of local disease, as in subacute and chronic gastritis, in some 
cases of ulcer of the stomach or the duodenum, in incipient carcinoma, 
and in certain cases of dilatation and atony. The withdrawal of 
chlorides from the food will also lead to a diminished production of 
hydrochloric acid. 

Anachlorhydria. — Not many years ago it was thought that the 
absence of free hydrochloric acid was pathognomonic of carcinoma 
of the stomach. This view was soon abandoned, however, as it was 
shown that cases of carcinoma occur in which hydrochloric acid is 
not only present, but present in excessive amounts. This is true 
especially of those cases in which the malignant growth has started 
upon the base of an old ulcer. It is noteworthy, moreover, that in 
early cases of carcinoma, even in the absence of ulcer, hydrochloric 
acid may at times be demonstrable and then disappear for days and 
weeks. It was furthermore shown that anachlorhydria exists in 
almost all cases of advanced chronic gastritis, in pernicious anemia 
(gastric anadeny), and is a fairly common occurrence in neurasthenic 
and hysterical individuals. In these cases periods of ana- hyper- 
and hypochlorhydria may alternate apparently without cause. In 
the acute febrile infections also anachlorhydria is not uncommon. 

Hyperchlorhydria. — Hyperchlorhydria (acid stomach, gastroxynsis) 
is very common in neurotic individuals, where it may alternate with 
hypo- and anachlorhydria. The same is seen even normally during 
menstruation. Associated with a continuous hypersecretion of gastric 
juice, it constitutes the neurosis known as hypersecretio acida et 
continua (gastrosuccorrhcea acida). Hyperchlorhydria is also of 
frequent occurrence in cases of gastric ulcer, and may even occur in 
carcinoma, notably in those cases in which, as stated above, the 
new-growth has started from an old ulcer. Regarding the frequency 
of hyperchlorhydria in ulcus there can be no doubt that this is found 



PLATE XII 




Fig. 1.— Congo-red Test. 
Fig. 2.— Dimethyl Reaction. 
Fig. S. — Alizarin Reaction. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 219 

in the majority of cases. Normal values, however, are by no means 
uncommon, and in some instances the amount of hydrochloric acid 
may be diminished. 

Hyperchlorhydria is also met with in passive congestion of the 
stomach (Schreiber's so-called "stagnant stomach"), in certain types 
of mental disease, in the early stages of chronic gastritis, during 
migraine attacks, etc. 

Test for Free Acids. The Congo-red Test. 1 — Congo-red is a car- 
mine-colored powder, while its solutions are of a peach- or brownish- 
red color, which changes to blue upon the addition of a free acid, but 
remains unaffected in the presence of an acid salt. Congo-red may 
be employed in solution or in the form of a test paper. The latter 
is less delicate than the solution, and indicates only the presence 
of 0.01 per cent, of hydrochloric acid, while a positive reaction can 
still be obtained with the aqueous solution in the presence of 0.0009 
per cent. The solution should be moderately dilute. The test paper 
is prepared by soaking filter paper, free from ash, in this solution, 
drying, and cutting it into suitable strips. In order to test for free 
acid, it is only necessary to immerse a strip of the test paper in the 
filtered gastric juice, or to add a drop or two of the solution to a small 
amount of the juice, when in the presence of a free acid a blue color 
will develop, which varies from sky-blue to a deep azure according 
to the amount present. (Plate XII, Fig. 1.) If the result is positive, 
the nature of the free acid must be ascertained, and it is, therefore, 
necessary to test for free hydrochloric acid, or in its absence for lactic 
acid and certain fatty acids. 

Tests for Free Hydrochloric Acid. — The various reagents which 
may be employed are given below, and are arranged according to their 
degree of delicacy, viz. : 

1. Dimethyl-amido-azo-benzol 0.02 pro mille. 

2. Phloroglucin-vanillin 0.05 " 

3. Resorcin 0.05 

4. Tropfpolin 00 0.30 

5. Mohr's reagent . 1.00 " 

The Dimethyl-amido-azo-benzol Test. 2 — This test has largely re- 
placed the older phloroglucin-vanillin and resorcin tests in the 
routine work of the clinical laboratory. The delicacy of the reagent 
is such that the natural yellow color of the indicator is changed to a 
reddish tinge upon the addition of but 1 drop of a one-tenth normal 
solution of hydrochloric acid in 5 c.c. of distilled water. Its superior 
delicacy, as compared with the phloroglucin-vanillin and resorcin 
tests, is apparent from the fact that 5 c.c. of a 0.5 per cent, solution 

1 Riegel, Deutsch. med. Woch., 1886, No. 35; and Boas, Diagnostik u. Thera- 
pie d. Magenkrankheiten. 

2 Topfer, Zeit. f. phvsiol. Chem., 1894, vol. xix. Hari, Arch. f. Verdauungs- 
krank., vol. ii, pp. 182 and 332. 



220 THE GASTRIC JUICE AND GASTRIC CONTENTS 

of egg albumen, to which 6 drops of a one-tenth normal solution of 
hydrochloric acid have been added, still give a positive reaction with 
dimethyl-amido-azo-benzol, while the phloroglucin-vanillin and 
resorcin reactions are negative. Organic acids, including lactic 
acid, yield a red color only when present in amounts exceeding 0.5 
per cent. I have further ascertained that if albumoses are present, 
a cherry-red color is not obtained even though lactic acid be present to 
the extent of 1 per cent. Loosely combined hydrochloric acid and 
salts do not produce a red color. 

For practical purposes a 0.5 per cent, alcoholic solution is em- 
ployed; 1 or 2 drops of this are added to a small quantity of 
the filtered gastric contents; in the presence of free hydrochloric 
acid a beautiful cherry red develops at once, which varies in intensity 
with the amount of free acid present (Plate XII, Fig 2.) In the 
presence of organic acids an orange color is obtained. In watery 
solution the color is a greenish yellow and the fluid is distinctly 
fluorescent. 

I have used Topfer's test for many years and am well satisfied 
with the results. In teaching students it is well to show the color 
which one obtains with lactic acid in the presence of albumoses; 
confusion as to whether or not free hydrochloric acid is present will 
then not occur. 

The Phloroglucin-vanillin Test. 1 — The solution employed con- 
tains 2 grains of phloroglucin and 1 gram of vanillin, dissolved 
in 30 c.c. of absolute alcohol; a yellow color results, which gradu- 
ally turns a dark golden red, changing to brown when exposed to 
light. The solution should therefore be kept in a dark-colored 
bottle. Lenhartz suggests the use of separate solutions of phloro- 
glucin and vanillin, 1 or 2 drops of each being employed in the 
test. Boas recommends a solution of the phloroglucin and vanillin, 
in the proportions indicated in 100 grams of 80 per cent, alcohol, 
and claims that the reagent is then still more sensitive and more 
stable. If a few drops of gastric juice, or even of the unfiltered 
gastric contents, containing 0.05 per cent, or more of free hydro- 
chloric acid, are treated with the same number of drops of the reagent, 
no change in color results, but upon slow evaporation — boiling and 
rapid evaporation are to be avoided — a general rose tint or fine rose- 
colored lines develop, which are characteristic of the presence af the 
free acid. 

For practical purposes it is best to carry on this slow evaporation 
on a thin porcelain butter dish, the porcelain cover of a crucible, or 
in a small evaporating dish of the same material. The color obtained 
in the presence of free hydrochloric acid is a rose color in every in- 
stance, and varies in intensity with the amount of acid present. A 

1 Gimzburg, Centralbl. f. klin. Med., 1887, vol. viii, No 40. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 221 

brown, brownish-yellow, or brownish-red color always indicates that 
excessive heat has been applied or that free hydrochloric acid is 
absent. 

Organic acids do not produce the reaction, nor is it interfered 
with by their presence, or that of albumins, peptones, or acid salts. 

A phloroglucin-vanillin test paper, prepared by soaking strips of 
filter paper, free from ash, in the solution and drying them, may 
also be employed. If a strip of this is moistened with a drop of 
gastric juice and gently heated in a porcelain dish, the rose color 
will develop in the presence of free hydrochloric acid, and does not 
disappear upon the addition of ether. 

The Resorcin Test. 1 — The solution consists of 5 grams of 
resublimed resorcin and 3 grams of cane sugar dissolved in 100 
grams of 94. per cent, alcohol. It is equally as delicate as the 
phloroglucin-vanillin solution and has the advantage of greater 
stability: 5 or 6 drops of gastric juice are treated with 3 to 5 
drops of the reagent and slowly evaporated to dryness over a small 
flame, when a beautiful rose- or vermilion-red mirror will be obtained, 
which gradually fades on cooling. If the reagent is employed in 
the form of a test paper, a violet color at first develops, which upon 
the application of heat turns brick red and does not disappear on 
treatment with ether. 

The presence of acid salts, organic acids, albumins, or albumoses 
does not interfere with the reaction. 

The Tropseolin Test. 2 — Tropseolin 00, when employed according 
to the method suggested by Boas, is a very reliable reagent, indi- 
cating the presence of 0.2 to 0.3 pro mille of free hydrochloric acid: 
3 or 4 drops of a saturated alcoholic solution of tropseolin 00, 
which has a brownish-yellow color, are placed in a small porcelain 
dish or cover, and allowed to spread over the surface. A like amount 
of gastric juice is added and likewise allowed to flow over the surface 
of the dish; upon the application of gentle heat a beautiful lilac 
appears, which is said to be characteristic of free hydrochloric acid. 

A tropseolin test paper may also be prepared by soaking filter 
paper, free from ash, in the alcoholic solution, and then drying and 
cutting it into strips. A few drops of gastric juice containing free 
hydrochloric acid produce a more or less pronounced brown color 
upon this paper, which turns lilac or blue upon the application of 
gentle heat. Organic acids, when present in large amounts, likewise 
produce a brown color, but this disappears on heating, and a lilac or 
blue color does not result. 

For ordinary purposes this test is sufficient, and recourse need only 



1 Boas, Centralbl. f. klin. Med., 1888, vol. ix, No. 45. 

2 Ewald, Klinik. d. Verdauungskrank., Berlin, 1888, vol. ii; and Boas, Deutsch. 
med. Woch., 1877, vol. xiii, p. 852. 



222 THE GASTRIC JUICE AND GASTRIC CONTENTS 

be had to the more delicate reagents when a negative or a doubtful 
result is obtained. 

The Combined Hydrochloric Acid.— It has been pointed out else- 
where that hydrochloric acid will only appear in the free state after 
all basic affinities have been saturated. For this reason combined 
hydrochloric acid must of necessity be present after the administration 
of a test meal if free acid can be demonstrated. If the contents 
are withdrawn too early free acid will be absent, while hydrochloric 
acid in combined form may be present in normal amount, considering 
the stage of digestion. From the mere absence of free hydrochloric 
acid it is hence not justifiable to infer that no hydrochloric acid has 
been secreted. Under pathological conditions it may happen that 
while the stomach has lost the power to furnish a sufficient amount 
of hydrochloric acid to satisfy the albuminous affinities of a large 
meal and to subsequently appear in the free state, enough can be 
furnished to meet the demands of a small meal. In any case then, 
where free hydrochloric acid is not found, it is important to ascertain 
whether no hydrochloric acid at all has been secreted. To this end 
the method of Martius and Liittke may be employed (see below). 

Quantitative Estimation of the Hydrochloric Acid of the Gas- 
tric Juice. Tbpfer's Method. 1 — The free and combined hydrochloric 
acid is most conveniently estimated according to Topfer's method, 
which is both simple and sufficiently accurate for clinical purposes. 

In this method the total acidity (a) of a given amount of gastric 
juice — i. e., the acidity referable to the presence of free hydrochloric 
acid, combined hydrochloric acid, acid salts, and any organic acids 
that may be present — is first determined (lactic acid and the fatty 
acids, if present, need not be removed), using phenol phthalein as an 
indicator. This is followed by a determination of the acidity refer- 
able to free acids and acid salts in another sample of gastric juice 
(b), using alizarin (alizarin monosulphonate of sodium) as an indi- 
cator. As this does not react with loosely combined hydrochloric 
acid, the difference between a and b will indicate the amount of 
the latter. The free hydrochloric acid (c) finally is estimated with 
dimethyl-amido-azo-benzol as an indicator, the difference between a 
and b-\-c giving the acidity referable to organic acids and acid salts. 

The solutions required are the following : 

1. A decinormal solution of sodium hydrate. 

2. A 1 per cent, alcoholic solution of phenolphthalein. 

3. A saturated aqueous solution of alizarin. 

4. A 0.5 per cent, alcoholic solution of dimethyl-amido-azo-benzol. 
Three separate portions of 5 or 10 c.c. of filtered gastric juice are 
measured into three small beakers or porcelain dishes. To the 
first portion 1 or 2 drops of phenolphthalein are added, when it 

1 Loc. cit. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 223 

is titrated with the one-tenth normal solution of sodium hydrate 
until a permanent pink color is obtained. 

To the second portion 3 or 4 drops of the alizarin solution are 
added, when it also is titrated with the one-tenth normal solution of 
sodium hydrate until a pure violet color is obtained (Plate XII, 

In the third portion the free hydrochloric acid is titrated, after 
the addition of 3 or 4 drops of the dimethyl-amido-azo-benzol, until 
the last trace of red — in the presence of free hydrochloric acid — has 
disappeared, and the color has become distinctly greenish yellow 
(Plate XII, Fig. 2). The results are then calculated as in the follow- 
ing example : 

10 c.c. of gastric juice, using phenolphthalein as an indicator, re- 
quired 6 c.c. of the one-tenth normal solution in order to bring about 
the end reaction, while a like amount titrated in the same manner with 
alizarin required 3 c.c. The difference between 6 and 3 indicates 
the number of cubic centimeters necessary to neutralize the amount of 
hydrochloric acid in combination with albuminous material. In the 
estimation of the free hydrochloric acid 2.3 c.c. of the one-tenth 
normal solution were required. 

The results can then be tabulated as follows: 

Total acidity (per 100 c.c. stomach contents) .... 60 
Alizarin acidity . . 30 

Combined hydrochloric acid ......... 30 

Free hydrochloric acid 23 

Total physiologically active hydrochloric acid .... 53 
Salts 7 

Total 60 

If not enough gastric juice is available for three separate titrations 
one can estimate the free hydrochloric acid in one portion of 5 c.c. 
with dimethyl as an indicator, and proceed at once to the total acidity 
in the same example. To this end phenolphthalein is added after the 
primary titration and the titration continued for the total acidity as 
usual. The first value will give the free hydrochloric acid and this 
plus the second value the total acidity. 

Deficit of Hydrochloric Acid. — When hydrochloric acid is absent 
it is customary to indicate the deficit in terms of — hydrochloric 
acid in a manner perfectly analogous to the method just now 
described, viz., 10 c.c. of gastric juice are treated with a few drops 
of dimethyl and then titrated with -f-^ hydrochloric acid until the red 
hydrochloric acid reaction appears. If 1 c.c. was necessary to this 
end the hydrochloric acid deficit would be 10. 

Estimation of Free Hydrochloric Acid (according to Sahli). — 25 to 30 
drops of Giinzburg's reagent are added to 10 c.c. of gastric juice. 



224 THE GASTRIC JUICE AND GASTRIC CONTENTS 

The mixture is titrated with a decinormal sodium hydrate solution 
as usual until a drop of the mixture, warmed on the stirring rod 
after each addition of the alkali, shows a red color. The rod must be 
washed and cooled after every test. 

The Method of Martius and Liittke (modified). 1 — This method 
is equally exact, but requires a greater expenditure of time. It is 
based upon the fact that upon incineration of the gastric juice the 
free hydrochloric acid and that loosely combined with albuminous 
material escape, while the chlorine in combination with inorganic 
bases remains in the mineral ash unless a very intense heat is ap- 
plied for some time. By subtracting the amount of chlorine present 
in the latter form from the total amount, the quantity in combina- 
tion with albuminous material and that occurring as free acid will 
be found. The total acidity of the gastric juice is then determined, 
and that referable to the presence of the free and combined hydro- 
chloric acid subtracted, the difference giving the amount of organic 
acids and acid salts. By determining the acidity due to the presence 
of free hydrochloric acid according to Topfer's method, and deducting 
the amount found from that referable to the presence of free and 
combined hydrochloric acid, the amount of the latter is obtained. 

Reagents required : 

1. A solution of silver nitrate in nitric acid of such strength that 
1 c.c. shall represent 0.00365 gram of hydrochloric acid. 

2. Liquor ferri sulphurati oxydati. 

3. A decinormal solution of ammonium sulphocyanide. 

4. A one- tenth normal solution of sodium hydrate. 

5. A 1 per cent, alcoholic solution of phenolphthalein. 

6. A 0.5 per cent, alcoholic solution of dimethyl-amido-azo-benzol. 
Preparation of the solutions : 

1. The silver nitrate solution. As a solution is required of such 
strength that 1 c.c. shall be equivalent to 0.00365 gram of hydro- 
chloric acid, the amount of silver nitrate that must be dissolved in 
1000 c.c. of water is ascertained in the following manner: Since 
169.66 (molecular weight) parts by weight of silver nitrate combine 
with 36.5 parts of hydrochloric acid (molecular weight), the amount 
of silver nitrate required for each cubic centimeter is found from the 
equation 

169.66 : 36.5 : : x : 0.00365; 36.5 £ = 0.6192590; s = 0.0169. 

In 1 c.c. of the silver solution 0.0169 gram of silver nitrate must 
thus be present, or 16.9 grams in the liter. This quantity, or roughly 
17 grams, is weighed off and dissolved in 900 c.c. of a 25 per cent, 
solution of nitric acid. To this solution 50 c.c. of the liquor ferri 
sulphurati oxydati are added. The solution is then brought to the 

1 Die Magensaure des Menschen, Stuttgart, 1982. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 225 

proper strength by titration of a known number of cubic centi- 
meters of a one-tenth normal solution of hydrochloric acid and 
correcting as usual (see below). 

2. The ammonium sulphocyanide solution. A normal solution 
of ammonium sulphocyanide contains 75.98 grams (molecular 
weight) per liter, and a decinormal solution 7.598 grams. This 
quantity, or roughly 8 grams, is dissolved in about 900 c.c. of water 
and the solution brought to the proper strength by titrating a known 
number of cubic centimeters of the silver nitrate solution, when ach 
cubic centimeter should correspond to 1 c.c. of the silver solution 
— i. e., to 0.00365 gram of hydrochloric acid. It is corrected as 
described elsewhere (see below). 

Method. — 1. To determine the total amount of chlorine present: 
10 c.c. of filtered gastric juice — Martins and Liittke make use of 
the unfiltered gastric contents — are measured into a small flask 
bearing a 100 c.c. mark, and treated with an excess of the one-tenth 
normal solution of silver nitrate. Experience has shown that 20 c.c. 
are sufficient. The mixture is agitated and allowed to stand for ten 
minutes. Distilled water is then added to the 100 c.c. mark; the 
mixture is agitated once more and filtered through a dry filter into 
a dry beaker; 50 c.c. of the filtrate are titrated with the one- 
tenth normal solution of ammonium sulphocyanide until the blood- 
red color which appears upon the addition of every drop— due 
to the formation of ferric sulphocyanide — no longer disappears on 
stirring. By multiplying the number of cubic centimeters of the 
ammonium sulphocyanide solution used by 2 (the number of cubic 
centimeters that would have been necessary for the precipitation of 
the excess of silver in 100 c.c.) and deducting the result from the 
number of cubic centimeters of the one-tenth normal solution of 
silver nitrate employed, viz., 20, the number of cubic centimeters 
of the latter solution is found which was necessary to precipitate 
the chlorine in 10 c.c. of the gastric juice. As 1 c.c. of the solu- 
tion represents 0.00365 gram of hydrochloric acid, it is only 
necessary to multiply this figure by the number of cubic centimeters 
used in precipitation of the chlorine. The resulting value, T, ex- 
presses the total amount of chlorine present. 

As a general rule, it is not necessary to decolorize the gastric 
juice. If desired, however, 5 to 15 drops of a 5 per cent, solution 
of potassium permanganate may be added to the 10 c.c. employed, 
after the mixture has stood for ten minutes. 

2. Determination of the amount of chlorine in combination with 
inorganic bases, F: 10 c.c. of the filtered gastric juice are care- 
fully evaporated to dryness in a platinum crucible, on a water bath 
or upon a plate of asbestos, in order to avoid sputtering (as the 
heat applied in the process of incineration is not very intense, a 
porcelain crucible may also be employed). The residue is then care- 
15 



226 THE GASTRIC JUICE AND GASTRIC CONTENTS 

fully incinerated over an open flame, the process being carried only 
to the point where the organic ash no longer burns with a luminous 
flame. Intense heat should be avoided, as the chlorides are volatil- 
ized upon the application of red heat. On cooling, the ash is moist- 
ened with a few drops of distilled water and mixed with a stirring 
rod, when the residue is extracted in separate portions with 100 c.c. 
of hot distilled water and filtered. This amount is usually suffici- 
ent to dissolve all the chlorides present. If any doubt should exist, 
however, it is only necessary to add a drop of the silver solution 
to a few drops of the last portion of the filtrate: the formation of 
a cloud, referable to silver chloride, will necessitate still further 
washing. The whole filtrate is then treated with 10 c.c. of the one- 
tenth normal solution of silver nitrate, and the amount consumed 
in the precipitation of the chlorides determined by titration with 
the one-tenth normal solution of ammonium sulphocyanide, as de- 
scribed above. The hydrochloric acid present in combination with 
inorganic bases is thus determined. The difference between the 
amount of hydrochloric acid in combination with inorganic bases 
and the total amount of chlorine in terms of hydrochloric acid will 
then indicate the amounts of the free and of the combined hydro- 
chloric acid, which are termed L and C, respectively; hence T — F 
= L + C. 

3. The total acidity in terms of hydrochloric acid is further de- 
termined according to the method given elsewhere (see p. 220) and 
indicated by the letter A. The difference between the total acid- 
ity and the amount of free and combined hydrochloric acid will 
represent the amount of organic acids and acid salts, 0; hence 
0=A— (L + C). 

The free hydrochloric acid finally is determined according to the 
method of Topfer. The difference between the value thus found 
and that expressing the amount of free and combined hydrochloric 
acid will indicate the amount of the latter; hence (L-\- C) — L= C. 

Leo's Method. 1 — This method is based upon the observation that 
calcium carbonate combines with free and combined hydrochloric acid 
at ordinary temperatures to form neutral calcium chloride, while the 
acid phosphates are not affected. It is thus clear that by determin- 
ing the total acidity of the gastric juice, and deducting from this 
the acidity referable to acid salts, the amount of the physiologically 
active hydrochloric acid — i. e., of the free and combined hydrochloric 
acid — is obtained. 

As it has been shown that in the presence of calcium chloride 
(formed, as indicated above, upon the addition of calcium carbonate), 
owing to the formation of calcium monophosphate — CaHP0 4 , twice 
the quantity of sodium hydrate is taken up, it is necessary to make 

» Centralbl. f. d. med. Wiss,, 1889. vol. xxvii. p. 481, 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 227 

the first titration also after the addition of an excess of calcium 
chloride. 

Reagents required: 

1. A one-tenth normal solution of sodium hydrate. 

2. A 1 per cent, alcoholic solution of phenolphthalein. 

3. A concentrated solution of calcium chloride. 

4. Chemically pure calcium carbonate. The purity of the salt 
may be tested by stirring a small piece with water; the solution 
should not color red litmus paper blue. A solution of the salt in 
dilute hydrochloric acid should not yield a precipitate when treated 
with sulphuric acid. 

Method. — Organic acids that may be present are first removed 
by shaking with ether, 50 to 100 c.c. being required for each 10 c.c. 
of gastric juice. The total acidity of the gastric juice is then de- 
termined in 10 c.c. of the filtered liquid after the addition of 5 c.c. 
of the concentrated solution of calcium chloride, the result being 
termed A. 

The acidity referable to the presence of acid phosphates is deter- 
mined as follows:' 15 c.c. of filtered gastric juice are treated with a 
pinch of dry and chemically pure calcium carbonate; the mixture is 
thoroughly stirred, and passed at once through a dry filter; 10 
c.c. of the filtrate, from which the carbon dioxide is expelled by 
means of a current of air, are then treated with 5 c.c. of the calcium 
chloride solution and titrated as above, the resulting value being 
termed P. A — P is hence equivalent to L-\-C. The value of C can 
then be ascertained by determining the acidity referable to free 
hydrochloric acid according to Topfer's method, and deducting 
the value found from L-\-C. 

This method is sufficiently accurate for practical purposes, and has 
the advantage of not requiring the expenditure of much time. 



The Ferments of the Gastric Juice and their Zymogens. 

Normal gastric juice contains three ferments, viz., pepsin, chy- 
mosin, and lipase. 

Pepsin and Pepsinogen. — According to our present knowledge, 
the zymogen of pepsin, \iz., pepsinogen or propepsin, and not pepsin 
itself, is secreted by the chief cells of the fundus glands. It is trans- 
formed into the ferment proper by the hydrochloric acid of the gastric 
juice. 

This is not the place to enter into a detailed consideration of the 
various properties of pepsin, and it will suffice to say that the activity 
of the ferment is destroyed by even very dilute solutions of the alka- 
line carbonates. The same result is reached by exposing a watery 
solution of pepsin to a temperature of 70° C, while in a dry state 



228 THE GASTRIC JUICE AND GASTRIC CONTENTS 

a temperature of 100° C. will not destroy its activity; this is shown 
by the fact that a specimen of pepsin thus treated is, on cooling, still 
capable of digesting albumins in the presence of hydrochloric 
acid. 

While pepsin is capable of digesting albumins in the presence of 
other acids, viz., phosphoric, sulphuric, oxalic, acetic, lactic, and 
salicylic acids, the solutions must be stronger than in the case of 
hydrochloric acid. With lactic acid, for example, a satisfactory 
result is reached only with a concentration of from 12 to 18 pro mille, 
while of hydrochloric acid 2 to 4 pro mille are sufficient. Larger or 
smaller amounts do not act so promptly. 

Figures expressing the exact quantity of pepsin or of its zymogen 
are lacking, and inferences can hence only be drawn as to the physio- 
logical activity of the ferment from the rapidity with which given 
amounts of albuminous material are digested. This, however, 
depends to a large extent upon the nature and concentration of the 
free acid present. Under normal conditions 25 c.c. of gastric juice 
will dissolve 0.05 to 0.06 gram of serum albumin in one hour, the 
same amount of coagulated egg albumen in three' hours, and a like 
amount of fibrin in one hour and a half. 

As abnormalities in the circulation and innervation of the stomach 
apparently do not influence the production of pepsin, or rather of its 
zymogen, a diminution in the degree of peptic activity, or its total 
absence, may be referred directly to disease of the stomach itself, 
viz., its glandular apparatus. The determination of the presence or 
absence and relative amount of pepsin in the gastric juice hence 
furnishes more useful information than the recognition of the presence 
or absence of free hydrochloric acid. 

As pepsin is formed from pepsinogen through the agency of a free 
acid, its presence, in the absence of organic acids in notable quan- 
tities, indicates at once the presence of hydrochloric acid. It may 
be said, vice versa, that if free hydrochloric acid is present in the 
gastric juice pepsin also will be found. Should the zymogen alone 
be present, digestion will take place only upon the addition of an acid, 
while an absence of digestion upon the addition of hydrochloric acid 
indicates the absence of both pepsin and its zymogen. At times, 
though rarely, a "gastric juice" is met with which is capable of 
digesting albumin in the absence of hydrochloric acid, owing to the 
presence of regurgitated pancreatic juice. 

In the differential diagnosis of a chronic gastritis and a neurosis, 
or a dyspeptic condition referable to hyperemia of the gastric mucous 
membrane, the demonstration of zymogen in the absence of hydro- 
chloric acid may, at times, be very important, bearing in mind that 
circulatory and nervous disturbances apparently do not influence 
the production of pepsinogen. An entire absence of the latter would, 
of course, warrant the diagnosis of anadeny of the stomach. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 229 

Tests for Pepsin and Pepsinogen. Test for the Enzyme. — If 
the presence of free hydrochloric acid has previously been ascertained, 
25 c.c. of filtered gastric juice are set aside and kept at a tempera- 
ture of from 37° to 40° C, a bit of coagulated egg albumen, fibrin, 
or serum albumin being added. In order to permit of a comparison 
of results, the same amounts should always be taken; 0.05 to 0.06 
gram of egg albumen, as has been shown, ought, under physiolog- 
ical conditions, to be digested in three hours. 

Test for the Zymogen. — Should hydrochloric acid be absent 
the test is made in the same manner, after the addition of from 3 to 
5 drops of the officinal solution of hydrochloric acid to 25 c.c. of the 
filtrate. Under such conditions usually pepsinogen alone is found. 

Quantitative Estimation of Pepsin. — Accurate methods for the quan- 
titative estimation of pepsin are unknown, and relative values only 
can be obtained. 

Hammerschlag's Method. 1 — Two Esbach tubes (albuminimeters) 
are employed. Tube A is filled to the mark U with a mixture of 10 
c.c. of a 1 per cent, solution of egg albumen 2 in 0.4 per cent, of hydro- 
chloric acid and 5 c.c. of filtered gastric juice. The second tube, B, 
receives a mixture of the same solution and 5 c.c. of water. After the 
tubes have been kept in the thermostat for one hour at a temperature 
of 37° C. Esbach's reagent (see Urine) is added to each tube to the 
mark R. After standing for twenty-four hours the amount of precipi- 
tated albumen is read off in the two tubes. The difference indicates 
the amount of albumen which was digested; this raised to the square 
gives the corresponding amount of pepsin (which of course is merely 
relative). The method suffices for practical purposes. 

Mett's Method. — Satisfactory comparative results can also be ob- 
tained with the method suggested by Mett. Capillary glass tubes 
are prepared measuring from 1 to 2 mm. in diameter. They are 
filled with white of egg, closed at the ends with breadcrumbs and 
coagulated in boiling water. After five minutes they are dried and the 
ends closed with melted paraffin. In this form they can be kept, 
but before use they should be examined to see that the column of 
albumen has not shrunk from the sides. Any bubbles that may be 
present disappear after two days. When needed they are cut into 
pieces from 1 to 2 cm. long. The length of the column digested in a 
given length of time serves as a measure of the digestive power of the 
specimen examined. In practice this column should be measured in 
millimeters with the aid of a magnifying glass, or a low power of the 
microscope, using a stage micrometer. The calculation of the corre- 
sponding amount of ferment is based upon the law of Schutz and 
Borrissow, viz., that the corresponding amounts of ferment in two 
solutions bear the same ratio toward each other as the square of the 

1 Wien. med. Presse, 1894, vol. xxxv, p. 1654. 

2 The white of one egg diluted about 13 times will make a 1 per cent, solution. 



230 THE GASTRIC JUICE AND GASTRIC CONTENTS 

number of millimeters of the column of egg albumen which has been 
dissolved in the same length of time. Nirenstein and Schiff 1 have 
ascertained that the length of the digested cylinder of albumen is 
proportionate to the length of time that digestion goes on, pro- 
viding that the length of the cylinder does not exceed 7 mm. If it 
does exceed this, digestion proceeds more slowly. It is hence ad- 
visable in all cases to dilute the gastric juice. In this manner another 
difficulty also is obviated, viz., the antipeptic activity which is caused 
by certain substances which are normally present in solution (prod- 
ucts of digestion, sodium chloride). Nirenstein and Schiff ascer- 
tained that a sixteenfold dilution with -^ HC1 (0.18 per cent.) is 
sufficient and that this prevents the digestion of more than 3.6 mm. 
in twenty-four hours, which is a further condition to ensure reliable 
results. 

Method. — The gastric juice is obtained after giving Ewald's test 
breakfast. 1 c.c. of the filtered contents is diluted with 16 c.c. of 
~ HC1 ; into this solution 4 Mett's tubes are placed and the mixture is 
kept in the incubator for twenty-four hours. The columns of digested 
albumen are measured and the average ascertained; this in terms 
of millimeters raised to the square and multiplied by 16 (the degree 
of dilution) indicates the relative amount of pepsin. If the digested 
column measures more than 3.6 mm. the gastric juice must be diluted 
thirty-two times. 

The unit of measure is the amount of pepsin by which 1 mm. 
of albumen is digested in twenty-four hours, with an acidity of 0.18 
per cent. HC1. Nirenstein and Schiff in their series found variations 
from to 256 pepsin units. 

Quantitative Estimation of Pepsinogen. — In order to estimate the 
amount of pepsinogen both Hammerschlag's and Mett's method can 
be applied after rendering the gastric contents acid with hydrochloric 
acid to the extent of from 1 to 2 pro mille. 

The Milk-curdling Ferment and its Zymogen, viz., Chymosin 
(Rennin) and Chymosinogen. — The specific action of chymosin is 
exerted upon milk, or lime-containing solutions of casein, which are 
coagulated in neutral or feebly alkaline solutions. 

In this connection it is important to note that the addition of a 
few cubic centimeters of a solution of calcium chloride, or any other 
soluble lime salt, results in a transformation of the zymogen into the 
physiologically active ferment, and that hydrochloric acid, while it 
normally causes such transformation, is not absolutely necessary in 
the presence of calcium chloride. 

Under physiological conditions chymosin and its zymogen are 
always present in the gastric juice. In disease the inferences that 
may be drawn from a quantitative estimation of the ferment and its 

1 Arch. f. Verdauungsk., 1903, vol. viii. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 231 

zymogen have been formulated by Boas/ to whom we are indebted 
for much valuable information in this connection : 

1. Notwithstanding the absence of free hydrochloric acid, chymo- 
sin may be present, although in minimal traces — i. e., demonstrable 
with a dilution of from 1 to 10 to 1 to 20 (see method below). 

2. In the absence of free hydrochloric acid the zymogen may still 
be present in normal amounts — i. e., demonstrable with a dilution 
of from 1 to 100 to 1 to 150. The presence of the zymogen, especially 
when repeatedly observed, probably always permits of the conclusion 
that we are not dealing with an organic disease of the stomach, but 
with a neurosis or a hyperemic condition of the mucous membrane 
referable to disease of other organs. 

3. The zymogen may occur in moderately diminished amount, 50 
per cent, only being present. This is usually owing to the existence 
of a gastritis which has not reached its highest degree of severity. 
The nearer the amount of zymogen approaches the normal, the 
greater will be the probability of an ultimate recovery under suit- 
able treatment. 

4. The amount of the zymogen is greatly diminished (dilutions of 
1 to 10 to 1 to 25 yielding a negative result) or may be absent alto- 
gether. In cases of this kind a severe and usually incurable gas- 
tritis exists, either primary or occurring secondarily to carcinoma, 
amyloid degeneration, etc. 

5. In conditions 1, 2 and 3, the reestablishment of the secretion 
of hydrochloric acid may be attempted with some prospect of success 
by means of stimulating remedies. 

These conclusions are based upon the employment of Ewald's 
test breakfast, and cannot be applied to observations made after 
other test meals, without previous studies in this direction. 

Testing for the presence of chymosin and its zymogen is of decided 
value in cases in which alkaline material is vomited, and where we 
may be called upon to decide whether this contains constituents 
of the gastric juice or not. 

Tests for Chymosin and Chymosinogen. Test for the Enzyme. — 
5 to 10 c.c. of milk are treated with 3 to 5 drops of the filtered 
gastric juice and kept at a temperature of 37° to 40° C. for ten to 
fifteen minutes. If coagulation occurs during this time, it may be 
concluded that the enzyme is present. 

Test for the Zymogen. — The milk is treated with 10 c.c. of 
the filtered and feebly alkalinized gastric juice and with 2 or 3 c.c. 
of a 1 per cent, solution of calcium chloride. The mixture is kept 
at a temperature of from 37° to 40° C, when in the presence of 
the zymogen the formation of a thick cake of casein will occur within 
ten to fifteen minutes. 

' » Centralbl. f. d. med. Wiss., 1887, vol. xxv, p. 417; and Zeit f. klin. Med., 1888, 
vol. xiv, p. 240. See also J. Friedenwald, Med. News, 1895. 



232 THE GASTRIC JUICE AND GASTRIC CONTENTS 

Quantitative Estimation. Of the Enzyme. — The method is 
based upon the fact that on gradually diluting a specimen of gastric 
juice a point is finally reached at which a chymosin reaction can no 
longer be obtained, the value being, of course, a relative one. Under 
physiological conditions a positive reaction can still be observed with 
a degree of dilution varying between 1 to 30 and 1 to 40. 

The gastric juice is neutralized with a very dilute solution of 
sodium hydrate. Tubes are then prepared containing from 5 to 10 
c.c. of the gastric juice, diluted in the proportion of 1 to 10, 1 to 20, 
1 to 30, etc., to which an equal amount of neutral or amphoteric milk 
is added. The tubes, properly labelled, are kept at a temperature 
of from 37° to 40° C, and the degree of dilution noted at which 
coagulation still occurs. 

Of the Zymogen. — The gastric juice is rendered feebly alkaline 
and tubes are prepared containing equal amounts of milk and gastric 
juice, the latter variously diluted, as above directed; the examina- 
tion is then carried on in the same manner. Normally a positive 
reaction is obtained with a dilution varying between 1 to 150 and 
1 to 100. 

Lipase. — The presence of lipase as a normal constituent of the 
gastric juice has now been definitely established. Its demonstra- 
tion and quantitative estimation are described in the section on 
the Urine. It is essential that the examination be made after a 
thorough washing of the stomach and the administration of a test 
meal which is free from fat. 



Analysis of the Products of Albuminous Digestion. 

In order to separate the various products of digestion from each 
other the following procedure may be employed : 

The filtered gastric contents are carefully neutralized with a dilute 
solution of sodium hydrate, using litmus paper to determine the re- 
action; a small drop of the mixture is placed upon the paper from 
time to time during the addition of the sodium hydrate until no 
change in color is produced either on the red or the blue paper. If 
syntonin is present, it will be precipitated, and can be collected on a 
small filter. Upon the addition of an excess of dilute acid or an 
alkali this precipitate will again be dissolved. The filtrate is feebly 
acidified with dilute acetic acid, treated with an equal volume of a 
saturated solution of common salt, and brought to the boiling point. 
Any native albumin that may be present in solution is thus coagulated 
and can be filtered off on cooling. In the filtrate the albumoses and 
peptones remain. 

By one-half saturation of the filtrate with ammonium sulphate, 
viz., by adding an equal amount of a saturated solution of ammonium 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 233 

sulphate, the primary albumoses can be precipitated. If then the 
neutral filtrate is treated with one-half its volume of a saturated 
solution of ammonium sulphate, which will thus give a two-third 
total saturation, a portion of the deutero-albumoses (fraction A) 
separates out on standing. This is filtered off and the solution 
saturated with ammonium sulphate in substance; the deutero- 
fraction B is thus thrown down, and on acidifying the filtrate with 
one-tenth of its volume of a solution of sulphuric acid that has been 
saturated with ammonium sulphate, and of which 10 c.c. correspond 
in strength to 17 c.c. of a ~ solution of sodium hydrate, the last traces 
of deutero-albumoses (fraction 0) will separate out on standing. 

The filtrate contains the " peptones." To demonstrate these a 
2 per cent, solution of cupric sulphate is added drop by drop, when 
in the presence of peptones a rose- to a purplish-red color will 
develop. 1 

Tests for the Products of Carbohydrate Digestion. 

Starch may be recognized by the fact that it strikes a blue color 
with a solution of iodopotassic iodide, while the same solution gives 
a violet or mahogany brown with erythrodextrin. To this end it 
is only necessary to add a drop or two of Lugol's solution to a few 
cubic centimeters of the filtered gastric juice. The presence of 
achroodextrin may be inferred if no change in color occurs upon 
the addition of the reagent. 

Maltose and dextrose, which both react with Fehling's solution 
and undergo fermentation, differ from each other in the fact that 
the former does not reduce BarfoecVs reagent on boiling. This is 
prepared by adding 1 per cent, of acetic acid to a 0.5 to 4 per cent, 
solution of cupric acetate. The rotatory power of maltose is about 
three times as strong as that of dextrose; (a) D= 150.4, as com- 
pared with 52.5. 

Lactic Acid. 

Mode of Formation and Clinical Significance. — The normal 
occurrence of lactic acid in the stomach during digestion was until 
recently regarded as an established fact and generally ascribed to 
the action of lactic acid producing organisms which had been swal- 
lowed and which could exercise their activity so long as hydrochloric 
acid did not appear in the free state. 

Martius and Ltittke, however, employing the method already 
described, found "that the accurately determined curve of acidity 

1 For a more detailed account of the chemistry of digestion and the analysis of 
the resulting products, see C. E. Simon, Physiological Chemistry, Lea Bros. & Co. 



234 THE GASTRIC JUICE AND GASTRIC CONTENTS 

referable to hydrochloric acid coincided in all respects, even at the 
beginning of the process of digestion, with the curve referable to the 
total acidity," so that lactic acid as a physiological constituent could 
not have been present. The researches of Boas, 1 moreover, prove 
beyond a doubt that in physiological conditions no appreciable amounts 
of lactic acid are formed during the process of digestion, and that the 
lactic acid found after an ordinary meal has been introduced into 
the stomach as such. It is known that lactic acid is present in 
various kinds of bread and it is, hence, not permissible to make use 
of any test meal containing lactic acid when the question as to its 
formation in the stomach is to be considered. For these reasons 
Boas suggests the use of simple oatmeal soup to which salt only has 
been added. For practical purposes this is probably not always 
necessary, as the small amount of lactic acid found after Ewald's 
test breakfast may usually be disregarded; an increased amount 
can be referred directly to pathological conditions. 

The fact that the lactic acid disappears or is at least no longer 
demonstrable at the height of digestion may be due to its resorption 
on the one hand, or to an interference of the hydrochloric acid with 
the delicacy of the reagent usually employed — i. e., Uffelmann's 
reagent — on the other. 

Under pathological conditions notable amounts (1 to 4 pro mille) 
of lactic acid are met with when stagnation of the gastric contents 
occurs as a result of motor insufficiency, in the absence of or with a 
diminished secretion of hydrochloric acid. It is hence a common 
symptom of carcinoma of the stomach. 2 It was indeed at one time 
thought that carcinoma was the only disease in which a notable 
lactic acid production took place, but experience has shown that the 
same may occur in benign cases of pyloric stenosis and gastric in- 
sufficiency. Such findings, however, are uncommon, and a high 
lactic acid value may still be regarded as strongly suggestive of 
malignant disease and especially when repeatedly observed. Early 
in the disease it appears that periods of chlorhydria and lactic acid 
production may alternate and it is desirable that this phase of the 
problem more particularly receive attention. . 

In cases in which carcinoma has developed upon the basis of an 
old ulcer, lactic acid may be absent and hydrochloric acid present in 
increased amount. 

In every case in which lactic acid is found the stomach should be 
thoroughly washed out in the evening and no food allowed until the fol- 

1 "Ueber d. Vorkommen v. Milchsaure im gesunden u. kranken Magen," Zeit. 
f. klin. Med., 1894, vol. xxv, p. 285. 

2 J. H. de Jong, "Der Nachweis d. Milchsaure u, ihre klinische Bedeutung," 
Arch. f. Verdauungskrank., vol. ii, p. 53. J. Friedenwald, "The Significance of 
the Presence of Lactic Acid in the Stomach," N. Y. Med. Jour., 1895. Rosenheim 
u. Richter, "Ueber Milchsaurebildung im Magen," Zeit. f. klin. Med., vol. xxviii, 
p. 505. 



PLATE XIII 




Kelling's Test for Laetie Acid. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 235 

lowing morning. Boas' test meal is then given and the examination 
repeated. If the presence of lactic acid can thus be established on re- 
peated examination, even if a normal condition or hyperchlorhydria can 
be demonstrated in the interval, an exploratory incision is justifiable. 

It should, finally, be mentioned that only that form of lactic acid 
which results from fermentative processes is of interest in this con- 
nection, and not the sarcolactic acid contained in meat. For this 
reason the demonstration of lactic acid after a meal of meat is of no 
diagnostic significance, so far as the question of carcinoma goes. 

Kelling's Method 1 (Author's Modification). — This test is best per- 
formed in the following manner : A test tubef ul of water receives a 
drop or two of a moderately strong solution of the sesquichloride of 
iron, so that the liquid is barely colored. One half is then poured 
into a second tube and serves as control. A small amount of the 
gastric filtrate is added to the other specimen, when in the presence 
of lactic acid a distinct yellow develops at once, which appears the 
more marked when compared with the nearly colorless control. 
This test is very delicate and to be preferred to the older method 
of Uffelmann. (Plate XIII). 

Uffelmann's Test. 2 — Heretofore Uffelmann 's reagent was quite com- 
monly employed in testing for lactic acid, but everyone who has 
had occasion to make frequent use of this reagent in clinical work 
must have been struck with the uncertainty of the results so often 
obtained. In a large majority of the cases, particularly if Ewald's 
test breakfast is. employed, a characteristic reaction — i. e., the occur- 
rence of a lemon or canary-yellow color — is not seen, notwithstanding 
the presence of lactic acid, but a pale yellow, brownish, grayish 
white, or even gray color is obtained instead, often leaving in doubt 
whether lactic acid is present or not. Aside from doubtful results, 
the value of the test is greatly diminished by the fact that glucose, 
acid phosphates, butyric acid, and alcohol give the same reaction, 
and that in the presence of such amounts of hydrochloric acid as are 
found at the height of normal digestion lactic acid is not indicated 
by the reagent. All these difficulties have long been appreciated, 
and in order to obviate at least some of them it was proposed to apply 
the test to an aqueous solution of the ethereal extract of the gastric 
contents : 

To this end 5 or 10 c.c. of the filtered gastric juice are extracted 
by shaking with from 50 to 100 c.c. of neutral sulphuric ether 3 in a 

1 "Rhodan im Mageninhalt; Zugleich ein Beitrag z. Uffelmann 'schen Milch- 
saurereagens," Zeit. f. phvsiol. Chem., vol. xviii. 

2 Deutsch. Arch, f. klin Med., 1880, vol. xxvi; and Zeit. f. klin. Med., vol. 
viii, p. 392. 

3 If lactic acid is not present in the |ree state, but in combination with albumin 
(i. e., if the Congo-red test is negative), it is necessary to set it free by adding 
dilute hydrochloric acid until the Congo test is just positive, as the ether will 
otherwise not extract it. 



236 



THE GASTRIC JUICE AND GASTRIC CONTENTS 



stoppered separating funnel for about twenty or thirty minutes; the 
ethereal extract is then evaporated on a water bath or the ether 
distilled off (no flame). The residue is taken up with from 5 to 
10 c.c. of distilled water and tested as follows: 3 drops of a 
saturated aqueous solution of ferric chloride are mixed with 3 
drops of a concentrated solution of pure carbolic acid and diluted 
with water until an amethyst-blue color is obtained; to this solution 
a portion of the ethereal extract is added, when in the presence of 
only 0.1 per cent, of lactic acid a lemon or canary-yellow color is 
obtained. 

Strauss' Method. *• — Instead of evaporating the ether as in the 
above method, the ethereal extract may be directly 
examined by shaking with a freshly prepared solu- 
tion of ferric chloride, as suggested by Fleischer. 
Making use of this principle, Strauss has con- 
structed an apparatus (Fig. 67) which will be 
found very convenient, and which permits of 
roughly determining the amount of lactic acid 
present. The instrument is essentially a sepa- 
rating funnel of 30 c.c. capacity, bearing two 
marks, of which the one corresponds to 5 c.c, 
the other to 25 c.c. The apparatus is filled with 
gastric juice to the mark 5, when ether (free from 
alcohol) is added to the 25 cc. line. After shaking 
thoroughly, the separated liquids are allowed to 
escape by opening the stopcock until the 5 c.c. 
mark is reached. Distilled water is then added to 
the 25 mark, and the mixture treated with 2 drops 
of the officinal tincture of ferric chloride, diluted 
in the proportion of 1 to 10. Upon shaking, the 
water will assume an intensely green color if more 
than 1 pro mille of lactic acid is present, while a 
pale green is obtained in the presence of from 0.5 
to 1 pro mille. The tincture of iron should be 
kept in a dark-colored dropping bottle of about 
50 c.c. capacity. 

It will be observed that only large amounts of 
lactic acid, which alone are of importance from 
a diagnostic point of view, are indicated by the 
apparatus. Small amounts, as those introduced 
with Ewald's test breakfast, or referable to lactic acid fermentation 
in the mouth, are not indicated, so that confusion as to the presence 
or absence of the acid can never arise. 

Vournaso's Method (Modification of Croner and Conheim). — 
The method has the advantage that extraction with ether is not 

1 "Ueber eine Modifikation d. Uffelmann'schen Reaktion," Berlin, klin. Woch. T 
1895, No. 37. 



Fig. 67.— Strauss' 
apparatus for the ap- 
proximative estima- 
tion of lactic acid. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 237 

necessary. It is based upon the formation of an isonitril on trans- 
forming lactic acid to iodoform and treating with an amino base. 
The isonitril is readily recognized by its disagreeable odor. 2 grams 
of potassium iodide are dissolved in a few (not more than 5) c.c. of 
water and 1 gram of sublimed, pulverized iodine added. The resultant 
solution is filtered through asbestos or glass wool and diluted to 50 
c.c. with distilled water; 5 c.c. of aniline are finally added. The reagent 
is kept in a dark-colored bottle and must be shaken before using; it 
keeps for a number of months. A few c.c. of the stomach contents 
(diluted if necessary) are rendered strongly alkaline with 10 per cent, 
caustic alkali solution, boiled and treated with a few c.c. of the re- 
agent. In the presence of lactic acid the offensive odor of isonitril 
appears either at once or on heating. 

With a dilution of 0.0025 gram in 100 c.c. the odor is still discern- 
ible. 

Alcohol and acetone give the same reaction. 

The chemical process which takes place is represented by the 
equations : 

1. 2CH 3 .CHOH.COOH + 10NfiOH+12I = 4H.COONa+6HI+2CHI 3 +4H 2 O. 

Lactic acid. 

2. CHI 3 +3NaOH +C 6 H 5 .NH 2 = 3NaI +C 2 H 5 NC +3H 2 0. 

Iodoform. Isonitril. 

Boas' Method. 1 — In doubtful cases the following method may 
be employed, as with it, and following the exhibition of Boas' test 
meal, all possible errors can be avoided. The stomach must be washed 
perfectly clean before the test meal is introduced. 

Principle of the Method. — When a solution of lactic acid is treated 
with a strong oxidizing agent and heated, the lactic acid is decomposed 
into acetic aldehyde and formic acid, according to the equation 

CH 3 — CH(OH)— CO.OH = CH 3 .CHO 4- H.CO.OH. 

Lactic acid. Acetic aldehyde. Formic acid. 

Practically, then, the test for lactic acid resolves itself into a test for 
acetic aldehyde, which can readily be recognized by testing with 
various reagents, such as an alkaline solution of iodopotassic iodide, 
Nessler's reagent, and others. Nessler's reagent is prepared as fol- 
lows : 2 grams of potassium iodide are dissolved in 50 c.c. of water and 
treated with mercuric iodide while heating, until some of the latter 
remains undissolved. Upon cooling, the solution is diluted with 20 
c.c. of water; 2 parts of this solution are then treated with 3 
parts of a concentrated solution of potassium hydrate; any precipitate 
that may have formed is filtered off, and the reagent kept in a well- 
stoppered bottle. When aldehyde is added to such a solution a 
yellowish-red or red precipitate results, the exact color depending 

1 Deutsch. med, Woch., 1893, No. 39; and Munch, med. Woch., 1893, No. 43. 



238 THE GASTRIC JUICE AND GASTRIC CONTENTS 

upon the amount of aldehyde present; 1 part of the aldehyde may 
still be recognized when diluted with 40,000 parts of water. 

With an alkaline solution of iodopotassic iodide, aldehyde in a 
dilution of 1 to 20,000 will still produce a cloudiness, referable to the 
formation of iodoform, which is readily recognized by its character- 
istic odor (Lieben's test for acetone). 

Method. — The filtered gastric juice is tested for the presence of 
free acids with Congo-red. If present, from 10 to 20 c.c. are evapo- 
rated to a syrup on a water bath, after the addition of an excess 
of barium carbonate, while the latter is unnecessary in the absence 
of free acids. The syrup is treated with a few drops of phosphoric 
acid, and the carbon dioxide removed by bringing it to the boiling 
point once only, when it is allowed to cool and extracted with 100 
c.c. of neutral sulphuric ether (free from alcohol), by shaking for 
half an hour. The layer of ether is poured off after half an hour, 
the ether is evaporated (no flame), the residue taken up with 45 c.c. of 
water, shaken and filtered, and finally treated with 5 c.c. of sulphuric 
acid and a pinch of manganese dioxide in an Erlenmeyer flask. 
This is closed with a perforated stopper carrying a glass tube bent 
at an obtuse angle, the longer limb of which passes into a narrow 
glass cylinder containing from 5 to 10 c.c. of Nessler's reagent or a 
like quantity of an alkaline solution of iodopotassic iodide. If heat 
is now carefully applied, the aldehyde formed by the oxidation of 
the lactic acid with manganese dioxide and sulphuric acid passes 
over when the boiling point is reached, and causes the precipitation 
of yellowish-red aldehyde of mercury in the tube containing the 
Nessler reagent, or of iodoform if the alkaline solution of iodine is 
employed. 

Quantitative Estimation of Lactic Acid According to Boas' 
Method. 1 — The principle already set forth also applies to the quanti- 
tative estimation of lactic acid. 

Solutions required: 

1. A one-tenth normal solution of iodine. 

2. A one-tenth normal solution of sodium thiosulphate. 

3. Hydrochloric acid (sp. gr. 1.018). 

4. A potassium hydrate solution (56 to 1000). 

5. Starch solution. 
Preparation of these solutions: 

1. A normal solution of iodine should contain 126.53 (molecular 
weight of iodine) grams of iodine in the liter, and a one-tenth normal 
solution, hence 12.6 grains. In order to dissolve the iodine 25 grams of 
potassium iodide are dissolved in about 200 c.c. of distilled water, 
when the 12.6 grams of resublimed iodine are added. This solution 
is then diluted with distilled water to the 1000 c.c. mark, and requires 
no further correction. 

1 Loc. tit., p. 237. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 239 

2. The one-tenth normal solution of sodium thiosulphate is pre- 
pared as described in the chapter on Acetone (see Urine). When 
treated with 1 gram of ammonium carbonate pro liter it will retain 
its titre almost indefinitely. 

3. Preparation of the starch solution: 5 grams of starch are dis- 
solved in 900 c.c. of water by heating, when 10 grams of zinc chloride 
in 100 c.c. of water are added. 

Method. — 10 to 20 c.c. of the filtered gastric juice are first 
treated as indicated above, viz., evaporated to a syrup after the 
addition of barium carbonate if free acids are present. A few drops 
of phosphoric acid are added, the carbon dioxide driven off by boil- 
ing, and the residue extracted, on cooling, with 100 c.c. of ether 
free from alcohol; the ether is evaporated after separation, the residue 
taken up with 45 c.c. of distilled water, and treated with manganese 
dioxide and sulphuric acid. The flask is closed by a doubly per- 
forated stopper; through one aperture a bent tube passes to the dis- 
tilling apparatus, and a straight tube provided with a piece of rubber 
tubing, clamped off, through the other. The latter should dip well 
down into the liquid, and serves for passing a current of air through 
the solution when the distillation is completed. The mixture is dis- 
tilled until about four-fifths of the contents have passed over, excessive 
heat being carefully avoided, as otherwise the aldehyde will be decom- 
posed, according to the equations: 

1. CH 3 .— CH(OH) — CO.OH = CH 3 .CHO + HCOOH. 

Lactic acid. Aldehyde. Formic acid. 

2. CH 3 .CHO + HCOOH + 20 = CH H .COOH + C0 2 + H 2 0. 

Aldehyde. Formic acid. Acetic acid. 

To the distillate, which is best received in a high Erlenmeyer 
flask, well stoppered, 20 c.c. of the one-tenth normal solution of 
iodine are added mixed with 20 c.c. of the 5.6 per cent, solution of 
potassium hydrate. The mixture is shaken thoroughly and allowed 
to stand for a few minutes. In order to liberate the iodine not used 
in the reaction, 20 c.c. of hydrochloric acid are added, and the ex- 
cess of iodine determined by titration with the one-tenth normal solu- 
tion of sodium thiosulphate. The titration is carried almost to the 
point of decolorization, when a little starch solution is added; the 
mixture is then titrated until the blue color has disappeared. The 
number of cubic centimeters of the one-tenth normal solution em- 
ployed, viz., 20, minus the number of cubic centimeters of the one- 
tenth normal solution of sodium thiosulphate, will then indicate the 
number of cubic centimeters of the former required for the formation 
of iodoform, viz., the amount of lactic acid present in 10 or 20 c.c. 
of gastric juice, as the case may be. As 1 c.c. of the one-tenth nor- 
mal solution of iodine has been found to indicate the presence of 
Q. 003388 gram of lactic acid, it is only necessary to multiply the 



240 THE GASTRIC JUICE AND GASTRIC CONTENTS 

number of cubic centimeters used by this figure, and the result by 
10, in order to obtain the percentage. 

The method described is reliable and sufficiently accurate for clini- 
cal purposes. At the same time it may be said that no more time 
is required than in the ordinary quantitative estimation of sugar by 
means of Fehling's method, or of hydrochloric acid according to 
the method of Martius and Liittke. 

Boas' Rapid Method. — This method is less accurate than the 
preceding one, but may be advantageously employed in the absence 
of the various reagents necessary with the former. 10 c.c. of 
filtered gastric juice are treated with a few drops of dilute sulphuric 
acid, and the albumin present removed by heat. The filtrate is evapo- 
rated to a syrup on a water bath, water added to the original amount, 
and this again evaporated to a small volume, fatty acids being thereby 
removed. The lactic acid remaining is now extracted with ether 
(200 c.c. for every 10 c.c. of gastric juice); the ether is evaporated, 
the residue taken up with water and titrated with a one-tenth nor- 
mal solution of sodium hydrate, using phenolphthalein as an indi- 
cator. As 40 parts by weight of sodium hydrate (molecular weight) 
combine with 90 parts by weight of lactic acid (molecular weight), 
and as 1 c.c. of the one-tenth normal solution of sodium hydrate con- 
tains 0.004 gram of sodium hydrate, the corresponding amount of 
lactic acid is found from the equation: 40: 90: 0.004: x; 40 x= 0.360; 
#=0.009. The value of 1 c.c. of the one-tenth normal solution in 
terms of lactic acid is thus 0.009. By multiplying the number of 
cubic centimeters used by this figure, the amount of lactic acid pres- 
ent in 10 c.c. of gastric juice is ascertained. The result multiplied 
by 10 indicates the percentage. 



The Fatty Acids. 

Mode of Formation and Clinical Significance. — Unless much 
milk or carbohydrates have been ingested, fatty acids do not occur 
in the gastric contents under physiological conditions, and it would 
appear from the researches of Boas 1 that their formation is intimately 
associated with that of lactic acid. After the exhibition of his test 
meal he was unable to demonstrate their presence either in health 
or in various diseases of the stomach, such as chronic gastritis, atony 
or dilatation referable to benign causes, etc. In carcinoma, however, 
fatty acids, such as lactic acid, were quite constantly found. Fliigge 
has shown that butyric acid can be derived from lactic acid and 
that this is probably its usual source. 

Acetic acid fermentation presupposes the presence of alcohol, 

1 Loc. cit. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 241 

whether this is introduced into the stomach as such or whether it 
results from the action of yeast (Saccharomyces cerevisise) upon sugar. 
It is, hence, necessary, whenever acetic acid is met with in the 
gastric contents, to exclude the presence of alcohol introduced from 
without. Only then is it permissible to refer its presence to stagnation 
and advanced decomposition of carbohydrates. 

If the examination is confined to an analysis of the gastric contents 
obtained otherwise than after the exhibition of Boas' or Ewald's 
test meal, the diagnosis of pyloric stenosis with dilatation is prob- 
ably always justifiable in the presence of notable quantities of buty- 
ric acid and acetic acid, while the same after a previous washing out 
of the stomach and the exhibition of Boas' test meal would suggest 
carcinoma as the cause of the stenosis. 

That butyric acid may occur in the gastric contents when butter 
or fats in general have been ingested is, of course, not surprising, 
and its presence then should be looked upon as a physiological occur- 
rence. At the same time it should not be forgotten that butyric acid, 
just as lactic acid, may possibly have been formed in the mouth, and 
conclusions should, hence, only be drawn when such sources of error 
can be definitely excluded,' and the amount found exceeds mere traces. 

In conclusion it may be said that in disease butyric acid is far 
more frequently encountered in the gastric contents than acetic acid, 
but the significance of the two, if alcoholism can be excluded, is the 
same. 

Tests for Butyric Acid. — 1. Butyric acid can usually be recog- 
nized by its odor alone, which is that of rancid butter. If a more 
definite test is desired we may proceed as follows : 

2. 10 c.c. of filtered gastric juice are extracted with 50 c.c. of 
ether. The ether is evaporated and the residue taken up with a few 
cubic centimeters of water. If a trace of calcium chloride in sub- 
stance is now added the butyric acid will separate out in the form of 
oil droplets, the nature of which is readily recognized by the pungent 
odor. If, instead of adding calcium chloride, a slight excess of 
baryta-water is used, strongly refractive rhombic plates or granular, 
wart-like masses of barium butyrate are obtained upon evaporation. 

3. Butyric acid may also be recognized by the peculiar odor 
of pineapple which develops when the dry residue of the ethereal 
solution is treated with a little sulphuric acid and alcohol. The 
reaction is due to the formation of ethyl butyrate (pineapple test). 

Tests for Acetic Acid. — 1. Like butyric acid, acetic acid can 
usually be recognized by its odor. 

2. 10 c.c. of filtered gastric juice are extracted with ether. The 
ether is evaporated, the residue dissolved in a few drops of water, 
and neutralized with a dilute solution of sodium hydrate, sodium 
acetate being formed. If to this a drop or two of a very dilute 
solution of ferric chloride is added, a dark-red color results. With 
16 



242 THE GASTRIC JUICE AND GASTRIC CONTENTS 

silver nitrate a precipitate is obtained which is soluble in hot 
water. 

Quantitative Estimation of the Fatty Acids. Method of Cahn- 
Mehring, Modified by McNaught. 1 — The total acidity is determined in 
10 c.c. of filtered gastric juice. Another 10 c.c. are evaporated 
to a syrup, diluted with water, and similarly titrated. • The difference 
between the two results will indicate the amount of fatty acids present. 



Gases. 

The stomach always contains a certain quantity of gases which 
have partly been swallowed and partly have passed into the stomach 
from the duodenum. As fermentative processes in health occur only 
when carbohydrates or fats have been ingested, and then only to a 
slight degree, nitrogen, oxygen, and carbon dioxide are the only 
gases found during the process of albuminous digestion. As the 
oxygen swallowed is, moreover, largely absorbed by the blood, and 
two volumes of carbon dioxide are returned for one volume of oxy- 
gen, the presence of large amounts of the former and small amounts 
of the latter is readily explained. In an analysis of the gases con- 
tained in the stomach of a dog which had been fed on meat, Planer 
found the following proportions: 

Carbon dioxide 25 . 2 vol. per cent. 

Oxygen 6.1 " " 

Nitrogen 68.7 " 

With a strict vegetable diet, on the other hand, hydrogen may 
also be found (Planer) : 

Man. Dog. 

Carbon dioxide . .20.79 33.83 32.9 vol. per cent 

Oxygen 0.37 0.8 " 

Nitrogen . . . .72.50 38.22 66.3 " 

Hydrogen . . . 6.71 27.58 

Marsh gas, CH 4 , a product of the fermentation of cellulose, may 
also be found in pathological conditions. It is yet an open question 
whether marsh gas is formed in the stomach or passes into the stomach 
from the small intestine. 

Such observations must, however, be regarded as rarities. In 
one case of this kind, examined by Ewald and Ruppstein, 2 in which 
alcohol, acetic acid, lactic acid, and butyric acid were found in the 
vomited material, an analysis of the gases gave the following result: 

1 Cited by Boas, Diagnostik u. Therapie d. Magenkrankheiten, 2d ed., 1891, 
p. 140. 

2 Ewald, Arch. f. Anat. u. Physiol., 1874, p. 217. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 243 

Carbon dioxide 20.6 vol. per cent. 

Oxvgen 6.5 " 

Nitrogen 41.4 " 

Hydrogen 20.6 " 

Marsh gas 10.8 " 

Traces of olefiant gas and of hydrogen sulphide were also found. 
It is curious to note that in this case the patient, who, according to 
his own statement, had a "vinegar factory in his stomach on one 
day and gas works on another day," was occasionally able to light 
the eructated gas at the end of a cigar-holder, where it burnt with 
a faintly luminous flame. McNaught has reported a similar case 
in which the analysis furnished the following results: carbon di- 
oxide, 56 per cent.; hydrogen, 28 per cent.; marsh gas, 6.8 per 
cent.; atmospheric air, 9.2 per cent. 1 

Ammonia and hydrogen sulphide are also at times met with; 
their presence is always due to albuminous putrefaction. 

Boas 2 found that hydrogen sulphide is quite commonly present 
in cases of dilatation referable to benign causes, while it is almost 
always absent in carcinoma. He adds that it is never found when 
lactic acid is present. In acute gastritis it may be observed tem- 
porarily. In a number of cases of carcinoma I have never found 
hydrogen sulphide. In one case reported by Strauss the Bacillus 
coli communis was apparently concerned in its production. 

To obtain a knowledge of the gases formed in the stomach during 
the process of digestion it is only necessary to fill an ordinary Doremus 
ureometer, or an Einhorn saecharimeter, with the unfiltered gastric 
contents, and to keep it at a temperature of from 37° to 40° C, 
when the evolution of gas can be followed closely and the necessary 
tests made. The presence of carbon dioxide is readily recognized 
by passing a small amount of sodium hydrate, in concentrated 
solution or in substance, into the tube, after the evolution has entirely 
ceased, when the fluid will rise. If other gases are present at the 
same time, they will remain after the carbon dioxide has been ab- 
sorbed. Hydrogen sulphide is readily recognized by its odor and 
by the fact that it will color a piece of filter paper, moistened with 
a few drops of sodium hydrate and lead acetate, a more or less pro- 
nounced brown or black. The test is conveniently made by filling 
a test-tube about half-full with the gastric contents and closing 
it with a cork stopper to which a strip of lead paper, prepared as 
indicated, is fastened. 



1 Kuhn, " Ueber Hefegahrung und Bildung brennbarer Gase im menschlichen 
Magen," Zeit. f. klin. Med., vol. xxi; and Deutsch. med. Woch., 1892, No. 49, 
and 1893, No. 15. 

2 "Ueber Schwefelwasserstoffbildung im Masenkrankheiten," Centralbl. f. inn. 
Med., 1895, No. 3; Deutsch. med. Woch., 1892, No. 49. Zawadzki, "Schwefel- 
wasserstoff im erweiterten Magen," Centralbl. f . inn. Med., 1894, No. 50. Dauber, 
" Schwefelwasserstoff im Magen," Arch. f. A 7 erdauungskrank., vol. iv, p. 4. 



244 THE GASTRIC JUICE AND GASTRIC CONTENTS 

Marsh gas is recognized by the fact that it burns with a scarcely 
luminous flame. 

The eructation of gas formed in the stomach should not be con- 
founded with the so-called eructatio nervosa, in which no gas is either 
eructated or air simply enters the esophagus and is expelled again 
with a loud, explosive noise. This may frequently be observed in 
neurasthenic and hysterical individuals, and is to a greater or less 
degree under the control of the will. 



Acetone. 

The presence of acetone in the gastric contents in pathological 
conditions has repeatedly been observed, especially by v. Jaksch and 
Lorenz, 1 and it is curious to note that the latter was at times able to 
demonstrate larger quantities of the substance in the gastric con- 
tents than in the urine. 

In the chapter on Acetonuria the relation existing between digest- 
ive diseases and the elimination of acetone will be dealt with more 
fully, but it may here be mentioned that in the primary diseases of 
the gastro-intestinal tract acteone is met with quite constantly in 
the gastric contents, while it is observed but rarely in the secondary 
forms, and never is seen in the gastric neuroses. This statement, 
however, is denied by SovelierT, who claims to have found traces of 
acetone in one case of nervous dyspepsia, while negative results 
were obtained in all other diseases of the stomach. I have repeat- 
edly been able to demonstrate the presence of acetone in cases of 
carcinoma, and never have found it in neurotic conditions. 

In order to test for acetone, the gastric contents are distilled after 
the previous addition of a small amount of phosphoric acid (1 to 
1000), when the tests of Reynolds and Gunning (see Urine) are 
applied to the distillate. If both reactions furnish a positive result 
the presence of acetone may be regarded as demonstrated. Den- 
niges' test may also be employed, and can be applied to the filtered 
contents directly (see Urine). 



Vomited Material. 

Food Material. — The vomiting of large amounts of totally undi- 
gested meat two or three hours after its ingestion is met with only 
in conditions associated with an entire absence of digestive juices 
from the stomach — i. e., in cases of atrophic cirrhosis of the stomach 
(anadeny of Ewald). This condition is not to be confounded with 

1 Zeit. f. klin. Med., 1891, vol. xix, p. 19. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 245 

the regurgitation of undigested food, mixed with mucus and saliva, 
which is seen in cases of stricture of the esophagus or of the car- 
diac orifice of the stomach. While at the outset of the latter dis- 
ease the regurgitation of food occurs immediately, or at least very 
soon, after a meal, it may take place between meals in the later 
stages of the disease when dilatation has occurred. The recog- 
nition of the origin of the material brought up may then be exceed- 
ingly difficult. In such cases an examination should be made for 
biliary coloring matter, which, if present, will, of course, immediately 
exclude the esophagus as the source of the material ejected. Un- 
fortunately, however, the reverse does not hold good. Small amounts 
of undigested meat are of no significance. The vomiting of well- 




Fig. 68. — Collective view of vomited matter. (Eye-piece III, objective 8 A, Reichert.) 
a, muscle fibers; b, white blood corpuscles; c, c', squamous epithelium; c", columnar epithe- 
lium; d, starch grains, mostly changed by the action of the digestive juices; e, fat globules; 
f, sarcinse ventriculi; g, yeast fungi; h, forms resembling the comma bacillus found by the 
author once in the vomit of intestinal obstruction; i, various microorganisms, such as 
bacilli and micrococci; k, fat needles, between them connective tissue derived from the 
food; I, vegetable cells, (v. Jaksch.j 

digested food is observed in some of the neuroses of the stomach, 
and also in certain cases of acute and subacute gastritis, ulcer of the 
stomach, and chronic gastritis in its early stages. The vomiting 
referable to cerebral and spinal diseases also belongs to this cate- 
gory. In this connection it is very important to enquire into the 
existence of nausea previous to the vomiting, for, as is well known, 
considerable amounts of saliva and mucus may be swallowed if 
much nausea has existed, the result being that the process of diges- 
tion is arrested before the occurrence of vomiting. In such an event 
it would be erroneous to conclude that, because the material ingested 
has not reached that stage of digestion which would be expected 
at the time of the vomiting, the stomach is incapable of properly 
performing its functions. 



246 THE GASTRIC JUICE AND GASTRIC CONTENTS 

Mucus. — The constant presence of large amounts of mucus in 
the gastric contents obtained with the stomach tube is almost path- 
ognomonic of the mucous form of gastritis, while its presence in 
vomited matter may be referable to preexisting nausea. In cases of 
pharyngitis moderate amounts of mucus are frequently found. The 
vomiting of pure mucus, according to Boas, is always pathognomonic 
of the absence of dilatation of the stomach, a statement founded on 
reason, as it is altogether unlikely that no particles of food should 
be brought up at the same time. 

Under the term gastrosuccorrhea mucosa Dauber 1 has described 
a condition in which large amounts of mucus are secreted by the 
non-digesting organ, in the absence of symptoms pointing to a gas- 
tritis. I have observed a similar case occurring in a neurasthenic 
patient, in which enormous quantities of mucus could at times 
be obtained from the fasting organ, but never during the process of 
digestion. A mild degree of hyperchlorhydria existed at the same 
time, as well as enteritis mucosa and rhinitis mucosa. The motor 
power was practically normal. 

Mucus is readily recognized on simple inspection by its glossy 
appearance. Chemically, it is distinguished by its behavior toward 
acetic acid (see Urine). 

Saliva. — The vomiting of pure saliva in the morning upon rising 
is a fairly common symptom of chronic pharyngitis, which in turn 
frequently carries in its train a chronic gastritis; it constitutes the 
so-called vomitus matutinus. Saliva, like mucus, is, of course, 
always present in the gastric contents in small amounts. Larger 
amounts are usually referable to an increased secretion owing to the 
existence of nausea. Chemically, saliva is best recognized by test- 
ing for the presence of the sulphocyanides (see Saliva). 

Bile. — Bile is rarely observed in the gastric contents brought up 
by the stomach tube, but is frequently seen in vomited matter, of 
which it may be said to be a constant constituent whenever the 
vomiting has been very intense or frequently repeated. Its presence 
in the former case should always excite suspicion of the existence of 
stenosis of the descending or horizontal portion of the duodenum or 
the beginning of the jejunum. This diagnosis becomes the more 
probable the more constant its presence. 

Pancreatic Juice. — Mixed with the bile there is probably always 
present some pancreatic juice, and it has been suggested that 
the constant absence of this constituent, in the presence of bile, is 
strongly suggestive of pancreatic disease or of obstruction of the 
pancreatic duct (the ductus Wirsungianus). 

The demonstration of pancreatic juice in the stomach is possible 

1 "Ueber kontinuirliche Magen-Schleimsecretion," Arch. f. Verdauungskrank., 
vol. ii, p. 167. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE 247 

only if the reaction is neutral or alkaline, as the pancreatic trypsin is 
destroyed by pepsin-hydrochloric acid. If then hydrochloric acid is 
absent it is well to ensure a distinctly alkaline reaction by adding 
a little 1 per cent, solution of sodium carbonate; a flake of fibrin is 
added and the mixture placed in the incubator; if digestion takes 
place the presence of trypsin is established. The flakes of fibrin may 
be previously colored with a little Magdala red; as digestion takes 
place the red is liberated and colors the fluid. 

Blood. — The presence of unaltered blood in the gastric contents 
is usually recognized without difficulty. If the hemorrhage has taken 
place in the stomach the color usually is dark brown or black owing 
to the action of the gastric juice upon the hemoglobin. Blood that 
is bright red in color and frothy is generally referable to a pulmonary 
hemorrhage, but it may happen that such blood remains in the 
stomach for some time and may then also appear brown or black. 
In the event of a large gastric hemorrhage, on the other hand, the 
blood may be vomited bright red in appearance. 

In order to recognize mere traces when the macroscopic and 
even the microscopic examination do not point to the presence 
of blood, the method of Miiller and Weber or that of Donogany 
should be employed. Kuttner claims that he was thus able to demon- 
strate the presence of blood in numerous cases of chlorosis in which 
other tests furnished negative results. I have been less successful 
in the disease in question, but admit that in cases of carcinoma and 
ulcer of the stomach it is with this method often possible to find 
traces of blood which would otherwise have remained unnoticed. 

The recognition of such "occult" bleeding is at times of great 
value in diagnosis. (See Occult Blood in the Feces.) 

Method of Miiller and Weber. — The gastric contents are treated 
with a few cubic centimeters of strong acetic acid and extracted with 
ether. Should the ether not separate in a clear layer after a few 
minutes, a few drops of alcohol are added. If the ether then re- 
mains colorless, no blood pigment is present, while a brownish- 
red color indicates the presence of acetate of hematin. As a similar 
but yellowish-brown and much less intense discoloration of the 
ether may be produced by other pigments, such as biliary coloring 
matter, it is well, in doubtful cases, to test the ethereal extract with 
guaiacum or aloin. (See Tests for Occult Blood in the Feces.) Spec- 
troscopic examination of the ethereal extract may also be resorted to. 
In the presence of blood an absorption band will be observed at the 
junction of the red and yellow. 

Donogany's Method. — A small amount of the suspected material 
is extracted with a 20 per cent, solution of sodium hydrate and 
filtered. A drop of the filtrate is then mixed on a slide with a drop 
of pyridin and covered with a cover-glass, when, in the presence of 
blood, orange-red crystals of hemochromogen will separate out on 






248 THE GASTRIC JUICE AND GASTRIC CONTENTS 

standing for a few hours. On spectroscopic examination these 
crystals will show the characteristic band of absorption between the 
yellow and the green. 

Hemorrhage from the stomach, hematemesis , may be observed in 
the most diverse conditions. It is either dependent upon a primary 
disease of the organ, such as ulcer and carcinoma, or it occurs sec- 
ondarily to disease of other organs, leading to a hyperemic condition 
of the gastric mucosa, such as the various forms of cardiac, renal, 
and hepatic disease, in connection with menstrual abnormalities, 
etc. In melena, purpura hemorrhagica, pernicious anemia, etc., the 
cause of the hemorrhage cannot always be determined. Nervous 
influences also may take part in the causation of gastric hemorrhage. 

Pus. — The occurrence of pus in vomited matter, referable to 
disease of the stomach itself, is uncommon. It is seen practically 
only in cases of phlegmonous and diphtheritic gastritis, and, as 
Strauss 1 has pointed out, in carcinoma affecting the smaller curva- 
ture and the region of the fundus. In such cases it is not uncom- 
mon to obtain as much as one-half to two tablespoonfuls of a muco- 
purulent fluid from the non-digesting organ. As the motor function 
in this form of carcinoma is often unimparied, the symptom may be 
of considerable value in diagnosis. The presence of larger quantities 
usually indicates perforation into the stomach of an accumulation of 
pus from a neighboring organ. An abscess of the liver, a suppurative 
pancreatitis, an abscess of the colon, or a subphrenic abscess may 
thus prove to be its primary source. When present in considerable 
amount pus is, of course, readily detected with the naked eye; if any 
doubt should arise, a microscopic examination will determine the 
question. 

Stercoraceous Material. — Very important from a clinical stand- 
point is the vomiting of stercoraceous matter which is notably observed 
in cases of ileus. Usually this is recognized without difficulty by its 
odor, which is referable to the presence of skatol. If any doubt 
should arise, it is only necessary to distil the vomited matter after the 
addition of a little phosphoric acid, and to test for the presence of 
phenol, indol, and skatol in the distillate, as described in the chapter 
on Feces. When chiefly derived from the small intestine, the vomited 
matter, according to v. Jaksch, will contain bile acids and bile pig- 
ment together with an abundance of fat, which may be detected 
by chemical or microscopic examination. The reaction is usually 
alkaline or feebly acid. 

I have had occasion to examine the vomited matter of a patient 
in whom an almost complete obstruction existed immediately above 
the ileocecal valve ; the color of the material was a golden yellow, the 
reaction neutral; no bile pigment or biliary acids were found, while 
hydrobilirubin was present. 

1 " Ueber Eiter im Magen," Berlin, klin. Woch., 1899, p. 870. 



MICROSCOPIC EXAMINATION OF GASTRIC CONTENTS 249 

Parasites. — Of parasites, ascarides, segments of teniae, trichinae, 
Ankylostoma duodenale, and Oxyuris vermicularis are, at times, 
encountered. Protozoa have been described in the stomach contents 
of patients with carcinoma, by Hensen, Striibe, Zabel, Ullmann, 
Cohnheim, Nichols, and others. (See Microscopic Examination of 
Stomach Contents.) 

Odor. — The odor of normal gastric juice is peculiar, suggesting 
the presence of an acid, which can be sharply distinguished from 
acetic or butyric acid. If blood is present in large amount, the 
vomitus emits an odor which is perfectly characteristic. A feculent 
odor is met with in cases of enterostenosis or in the presence of an 
abnormal communication between the stomach and the small or 
large intestine. A putrid odor may be observed in cases of ulcera- 
tive carcinoma, pyloric stenosis referable to ulcer, simple carcinoma 
of the stomach, muscular hypertrophy of the pylorus, stenosis due 
to inflammatory adhesions, etc. In cases of phosphorus poisoning 
the vomited matter emits an odor of garlic; the odor observed in 
uremic conditions is referable to ammonia; a carbolic acid odor is met 
with in cases of poisoning with this substance. 



MICROSCOPIC EXAMINATION OF THE GASTRIC CONTENTS. 

In the gastric contents obtained from the non-digesting stomach 
the various morphological constituents of ihucus and saliva, which 
have been described elsewhere, are found. Microscopic particles of 
food, such as elastic tissue fibers, starch granules, fat droplets, fatty 
acid crystals, vegetable and muscle fibers, are, furthermore, quite 
constantly seen. Leukocytes and isolated nuclei also are observed; 
the latter are set free by the action of the gastric juice upon the 
mucous corpuscles and epithelial cells. 

If gastric juice is allowed to stand, small tapioca-like bodies will 
collect at the bottom of the vessel, which upon microscopic exami- 
nation will be seen to contain numerous snail-shell-like formations, 
occurring either singly or collected in groups. These probably con- 
sist of altered mucin, as they can be produced artificially by adding 
a sufficient amount of dilute hydrochloric acid to saliva. Accord- 
ing to Boas, they are of no diagnostic significance. 

Epithelial cells, fragments of the epithelial lining of the ducts of 
glands, as well as goblet cells, are not infrequently met with in the 
juice obtained from the non-digesting organ, In addition, various 
microorganisms, such as the Leptothrix buccalis, Bacillus subtilis, 
saccharomyces, micrococci (often arranged in the form of tetrahedra), 
Clostridium butyricum, etc., may be encountered. 

Among the bacteria which may be found in the gastric contents 
under pathological conditions the bacillus described by Boas and 



250 THE GASTRIC JUICE AND GASTRIC CONTENTS 

Oppler 1 is undoubtedly the most important, and has attracted much 
attention. It is quite constantly present in carcinoma, at a time when 
lactic acid can be demonstrated in large amount. It is an active 
lactic acid producer and its presence may hence be regarded as indi- 
cating advanced lactic acid fermentation. It is almost always absent 
in non-malignant disease of the stomach. The organism (Fig. 69) 
is non-motile, and essentially characterized by its great length and 
by the fact that the individual bacilli are frequently seen joined end 
to end, forming long threads and zigzag lines. Often the entire field 
of vision is filled with dense conglomerations, and in advanced cases 
it is usual to find the Boas-Oppler bacillus present almost exclusively 
in viable form. The organism is readily stained with the usual 
aniline dyes. I have succeeded in growing the organism on blood 
serum and usually also on plain agar, but it is very apt to undergo 
changes in size which may lead one to think that it has been lost or 







Fig. 69. — Boas-Oppler bacillus. 

overgrown by other bacilli. Growth may sometimes be obtained by 
rendering the culture medium acid with lactic acid to the extent to 
which this was present in the stomach contents. 

Tubercle bacilli may be found in vomited matter in cases of phthisis, 
where the sputa have been swallowed. Tuberculous ulceration of the 
stomach is exceedingly rare. Simmonds reports that in 2000 autopsies 
of tuberculous individuals the condition was noted only eight times. 

Sarcince (see Fig. 68) occur in the form of peculiar colonies of cocci, 
arranged in squares or tetrahedra, resembling cotton bales. Not 
infrequently they are encountered under normal conditions, but only 
in small numbers. In pathological conditions, on the other hand, a 

1 " Zur Kenntniss des Mageninhalts bei Carcinoma ventriculi," Deutsch. med. 
Woch., 1895, Nr. 5. Kauffmann, " Ueber einen neuen Milchsaurebacillus," etc., 
Wien. klin. Woch., 1895, Nr. 8. Schlesinger u. Kauffmann, Wien. klin. Rund- 
schau, 1895, Nr. 15. 



MICROSCOPIC EXAMINATION OF GASTRIC CONTENTS 251 

drop of the gastric contents may constitute an almost pure culture. 
A case is on record in which the pylorus had become entirely occluded 
by an inspissated mass of these organisms. Whenever present the 
existence of certain fermentative processes may be inferred. It is 
curious to note that in advanced cases of carcinoma of the stomach 
sarcinse are practically never seen, although the conditions are appa- 
rently most favorable for their development. Oppler 1 was unable 
to find them twenty-four hours after their introduction in large 
numbers and in pure culture. In cases of carcinoma of the curva- 
tures and the walls, as also in advanced pyloric carcinoma, sarcinne 
were never found, while they may be present in incipient cases of 
pyloric carcinoma so long as hydrochloric acid is secreted. 

Protozoa have been found in the stomach contents by several 
observers. Nichols 2 has collected 23 cases from the literature. The 
most common are trichomonads and next in order Megastoma enteri- 
cum (Lamblia intestinalis) ; whether or not still other varieties occur 
is not clear from the meager descriptions which are usually given. 
Flagellates, amebas, and monads are mentioned in a general way. 
Megastoma and trichomonads may be found together. The presence 
of protozoa is most common in carcinoma of the stomach (19 out of 
23 cases). The reaction of the material in which they are found is 
almost invariably alkaline or neutral. It is noteworthy that in several 
cases trichomonads were also found in carious teeth and in many 
in the stools of the patients. 

In esophageal carcinoma protozoa have also been found in the 
esophageal material. 

From the available data there can be no question that the presence 
of protozoa in the stomach contents is suggestive of non-obstructive 
carcinoma. To hunt for the parasites it is best to obtain material 
from the fasting organ and to examine this as soon as possible, taking 
care that it is not exposed to cold. Attention should be especially 
directed to any solid particles that may be visible with the naked eye. 

In vomited material containing biliary coloring matter, leucin, 
tyrosin, and cholesterin are quite commonly observed, and may be 
recognized by the form of their crystals, as well as by their chemical 
reactions, which are described elsewhere. 

The occurrence of blood and pus in the gastric contents has been 
considered. 

It not infrequently happens that small shreds of mucous mem- 
brane are brought away by the stomach tube, and in cases of chronic 
gastritis, hyperchlorhydria not dependent upon ulcer, and in some 
of the neuroses, this is indeed not at all uncommon. 3 Boas even 

1 Munch, med. Woch., 1894, No. 29. 

2 Amer. Journ., July, 1905, p. 120. G. Striibe, "Trichomonas hominis bei 
Carcinoma ventriculi," Berlin, klin. Woch., 1898, p. 708. P. Cohnheim, Deutsch. 
med. Woch., 1903, vol. xxix, p. 206. 

3 M. Einhorn, Med. Record, June 23, 1894; Berlin, klin. Woch., 1895, No. 20; 
Arch. f. Verdauungskrankheiten, vol. v, Heft 3. 



252 THE GASTRIC JUICE AND GASTRIC CONTENTS 

suggests that in the neuroses, where fragments of mucous membrane 
are so readily detached, this may possibly be connected etiologically 
with the formation of ulcers, and there can be no doubt that the 
mere action of the abdominal muscles exerted during the process 
of defecation may be sufficient to detach such fragments. From 
the microscopic appearance of the particles the diagnosis between a 
gastric neurosis and one of the various forms of chronic gastritis 
may frequently be made, and the same may be said to hold good in 
the differential diagnosis between a true gastritis and a glandular 
insufficiency referable to passive congestion of the gastric mucosa. 

At times tumor particles also are found in the gastric contents. 1 
When particles of tissue are found they should be hardened at once, 
and then sectioned. 



EXAMINATION OF THE MOTOR POWER OF THE STOMACH. 

Under physiological conditions the stomach should contain but 
few particles of food, or none at all, six hours after the ingestion of 
Riegel's meal, or one and one-half to one and three-quarters hours 
after that of Ewald.- A delay in the propulsion of the gastric contents 
may be referable to the existence of a simple atony or to dilatation 
of the stomach. According to Boas, an atony may usually be diag- 
nosticated if, following the exhibition of a supper consisting of bread 
and butter, cold meat, and a large cupful of tea, the stomach is found 
empty in the morning, providing, of course, that symptoms exist 
which point to atony or dilatation. It should be remembered, however, 
that in cases of acute and subacute gastritis, in the absence of a more 
serious lesion, food may be found in the stomach twenty-four hours 
after its ingestion. A dilatation may, on the other hand, be diagnosti- 
cated if the stomach under the same conditions contains a considerable 
amount of food. In such cases it happens that not only remnants 
of the test supper, but remains of meals taken one, two, three, or 
even more days previously are found. The quantities, moreover, 
which may be obtained at the time of examination are often surpris- 
ingly great, and may amount to sixteen pounds or more. Portel 
cites the case of the Due de Chausnes, one of Paris' greatest gour- 
mands, whose stomach could hold 4.5 liters — i. e., 8 pints. 

The following methods may be employed for the purpose of testing 
the motor power of the stomach: 

Leube's Method. 2 — Six hours after the ingestion of Riegel's 
meal the stomach is washed out with about 1000 c.c. of water. In 
the presence of only slight traces of food the motor power may be 

1 P. Cohnheim, " D. Bedeutung kleiner Schleimhautstiickchen f . d. Diagnostik 
d. Magenkrankheiten," Arch. f. Verdauungskrankheiten, 1896, vol. i, p. 274. 

2 Deutsch. Arch. f. klin. Med., vol. xxxiii. 



EXAMINATION OF BESOBPTIVE POWER OF STOMACH 253 

regarded as normal. This method is undoubtedly the most con- 
venient for practical purposes. 

The Salol Test of Ewald and Sievers. 1 — This test is based upon 
the observation that salol is decomposed into phenol and salicylic 
acid only in an alkaline medium. As the salicylic acid is eliminated 
in the urine as salicyluric acid, it is possible to determine the time 
of the passage of the salol from the stomach to the small intestine. 

A capsule containing 1 gram of salol is given to the patient im- 
mediately after his breakfast or dinner, when separate portions of 
urine, passed one-half, one hour, two hours, and twenty-four hours 
later, are tested by adding a small amount of a solution of ferric 
chloride. In the presence of salicyluric acid a violet color results. 
Under normal conditions a positive reaction is obtained after from 
forty-five to seventy-five minutes. A further delay may usually be 
regarded as indicating the existence of motor insufficiency. If no 
result is obtained after twenty-four hours, a pyloric stenosis undoubt- 
edly exists. Under normal conditions, furthermore, it will be observed 
that the salol elimination is completed after twenty-four hours, 
while in cases of dilatation of the stomach a positive reaction may 
still be obtained after thirty hours. It is thus possible to distinguish 
between dilatation and descent of the stomach. 

The test, while it is convenient and usually yields fair results, is 
not altogether reliable, as the decomposition of the salol may at 
times occur in the stomach, owing to the presence of alkaline mucus, 
or may be delayed in the intestines owing to the existence of acid 
fermentation, etc. 2 



EXAMINATION OF THE RESORPTIVE POWER OF THE STOMACH. 

To this end a capsule containing 0.2 gram of potassium iodide 
is given to the patient shortly before a meal, and the saliva examined 
for the presence of potassium iodide at intervals of from two to three 
minutes. 3 To this end strips of filter paper moistened with starch 
solution are immersed in the saliva, which has been acidified with 
nitric acid; the paper turns blue if iodide be present. Under normal 
conditions a violet color is obtained after from six and one-half to 
eleven minutes, and a bluish tint after from seven and one-half to 
fifteen minutes. In pathological conditions a delayed reaction is 
observed in almost all diseases of the stomach, and is especially 
marked in cases of dilatation and carcinoma, less so in chronic gas- 
tritis, and variable in ulcer. 

1 Therap. Monats., August, 1887. 

2 Brainier, Deutsch. med. Woch., 1889. Huber, Correspondenzbl. f. schweizer 
Aerzte, 1890. 

3 Penzoldt, Berlin, klin. Woch., 1892 Faber, Inaug. Diss., Erlangen, 1882. 



254 THE GASTRIC JUICE AND GASTRIC CONTENTS 

Absolute conclusions, however, cannot be drawn from results thus 
obtained, as a normal reaction time has also been observed in cases 
of dilatation and chronic gastritis. 



INDIRECT EXAMINATION OF THE GASTRIC JUICE. 

Giinzburg' S Method. — In those cases in which for any reason the 
introduction of the stomach tube is contra-indicated or impracticable 
the following method, suggested by Giinzburg, may be employed: 

A tablet of 0.2 to 0.3 gram of potassium iodide is inserted into 
a piece of the thinnest possible, strongly vulcanized rubber tubing, 
measuring about 2.5 cm. in length. The ends are folded as shown 




Fig. 70. — A fibrin-potassium-iodide package of Giinzburg. 

in Fig. 70, and the little package tied with three threads of fibrin 
hardened in alcohol. Every package should be examined before 
use, by immersion in warm water for several hours, to determine its 
tightness, testing for the presence of potassium iodide by means of 
starch paper and fuming nitric acid. One of these packages is 
swallowed by the patient three-quarters to one hour after an Ewald 
test breakfast, and the saliva tested for potassium iodide at intervals 
of fifteen minutes, until a positive result is reached or until six hours 
have elapsed. It is unnecessary to wait longer than six hours. In 
the presence of free hydrochloric acid the threads of fibrin are dis- 
solved and the potassium iodide absorbed. Under normal conditions 
a positive reaction is obtained after from one to one and three-quar- 
ters hours, while anachlorhydria undoubtedly exists if no result is 
obtained within five or six hours. In cases of hypochlorhydria the 
reaction is delayed for more than two to three hours. Giinzburg 
further advises that the resorption test with potassium iodide be 
also made, and that the reaction time be deducted from that taken 
up in the elimination of the iodide contained in the package. Sev- 
eral tests, moreover, should be made in the same case. 

I have had occasion to experiment with packages obtained from 
Germany, and manufactured according to the directions of Giinz- 
burg. 1 In most of the packages the threads of fibrin had become 
brittle and were broken in transit. The results obtained with about 
twenty intact specimens, however, were entirely satisfactory, and it 
is to be regretted that the packages cannot be obtained in the American 
market. 

1 Gothe Apotheke, Frankfurt a. M. 



INDIRECT EXAMINATION OF THE GASTRIC JUICE 255 

Similar packages have been constructed by Sahli (desmoid reac- 
tion). In this case pills of methylene blue or iodoform are enclosed 
in little pieces of rubber tissue and closed with catgut. They are 
administered at the noon meal and the urine (viz., saliva) tested at 
5 and 7 p.m. and again in the morning. 1 

Reach has of late made use of barium iodate and the oxyiodate of 
bismuth for the same purpose, but without enclosing the substance 
in rubber. As hydrochloric acid only is capable of liberating the 
iodine from these bodies, they may be employed instead of the Giinz- 
burg packages. As a result of his examinations, he concludes that 
in the presence of hydrochloric acid iodine can thus be demonstrated 
in the saliva within eighty minutes. He finds, however, that at 
times the reaction occurs later than might have been supposedjrom 
the amount of hydrochloric acid found. 

1 Monod, Journ. de physiol. et de pathol. gen., 1906, vol. viii, p. 853. 



CHAPTER IV. 

THE FECES. 

The feces constitute a mixture of indigestible and undigested 
particles of food, of unabsorbed secretions of the gastro-intestinal 
tract, and their decomposition products, together with intestinal 
mucus, epithelial cells, and bacteria. 

EXAMINATION OF NORMAL FECES. 
General Characteristics. 

Number of Stools. — The number of stools which may be passed 
in the twenty-four hours is subject to wide variation, even under 
physiological conditions, but is usually constant for one and the same 
individual. One or two stools pro die may be regarded as normal. 
Exceptions, however, are frequent. Persons are thus met with who 
have but one stool every two to four days, and cases are on record 
in which only one passage occurred every seven to fourteen days, 
the individuals evidently enjoying perfect health. On the other 
hand, the number of stools may be increased to three or four under 
strictly normal conditions. Hence the importance of accurately ascer- 
taining the habitual number of stools in every individual. It would 
thus be manifestly wrong to regard the passage of three stools daily 
as diarrhea, or the passage of only one stool in forty-eight hours as 
constipation, if this number has been habitual throughout life. 

Diarrhea is said to exist when the consistence of the stools is 
materially diminished; the number is then also usually increased. 
This may vary from two to thirty, forty, and even fifty in the twenty- 
four hours. On the other hand, a single stool in the twenty-four 
hours may constitute diarrhea. The most extreme grades of diarrhea 
are observed in Asiatic cholera, dysentery, and the summer diarrhea 
of infants. 

Amount. — In those cases in which more than one or two stools 
occur in twenty-four hours it is well to ascertain the amount actually 
passed. The normal amount varies between 100 and 200 grams. 1 
This quantity is increased by a diet rich in vegetable and starchy 

1 Voit, Zeit. f. Biol., vol. xxv, p. 264. 



EXAMINATION OF NORMAL FECES 



257 



foods, and is diminished by one rich in animal proteids, so that 60 
and 270 grams may be regarded as the extreme limits in health. 
Such amounts as 500 and 1000 grams are certainly abnormal. 

Average quantities for various ages are given in the following 
table, which is taken from Schmidt and Strassburger i 1 



Child, 







Average amount of 


Age. 


Diet. 


feces in twenty-four 
hours. 


1 month old . . 


. . Mother's milk 


3.3 grams 


2 to 3 months old 


it it 


6.5 " 


7 


. . Variable 


15-56 " 


9 


. . Cows' milk with 






additions 


59.0 " 


f to 2 years old . 


. . . Mixed 


77.0 " 


4 " " . 


a 


101.0 " 


6 " " . 


a 


134.0 " 


9 " " . 


" 


117.0 " 


1 " " . 


it 


138.0 " 




it 


131.0 " 



Adult 

Unusually large amounts of fecal matter may be observed follow- 
ing an attack of constipation of long duration or an attack of obstruc- 
tion. Lynch reports a remarkable instance in which, following a 
prolonged attack of constipation, an enema caused the evacuation of 
20 kgrms. of fecal matter. Especially large amounts of feces are 
observed in cases of biliary obstruction, where 1100 grams may be 
exceeded. In cases of fermentative dyspepsia the amount may also 
be large, varying between 400 and 900 grams, while the patients are 
on a diet on which normal individuals would pass from 200 to 270 
grams in the twenty-four hours. Still larger amounts are noted in 
cases of enteritis. Schmidt mentions a case in which 2780 grams 
were eliminated (these figures have reference to a three days' experi- 
ment with a test diet; see p. 268). 

Consistence and Form.— The consistence of a stool depends 
essentially upon the amount of water present, and hence upon the 
nature of the food ingested, being softer with a purely vegetable 
diet (80 to 85 per cent, of water) than with a diet rich in animal pro- 
teids (60 to 65 per cent.). With a mixed diet the amount of water 
corresponds to about 75 per cent. As a general rule, normal stools 
exhibit the characteristic cylindrical form and are fairly firm. Mushy 
stools, however, are also seen quite frequently, and round, scybalous 
masses, although far more common in constipation, may likewise be 
observed in health. The individual scybala usually vary in size 
from that of a hazelnut to that of a walnut, and are frequently pro- 
vided with one or two indentations which represent impressions of 
the tenia of the colon. Still smaller masses, resembling the dejecta 
of sheep, may also be seen. Their presence was formerly regarded 
as characteristic of stricture of the colon, but they are likewise found 
in ordinary cases of chronic constipation. Fecal ribbons and columns 



Die Faeces d. Mehschen, Berlin, 1961, A. Hirschwald. 



17 



258 THE FECES 

of the diameter of a pencil are found in cases of enterospasm of neu- 
rotic origin, as well as in stricture of the colon. 

Odor. — The repugnant odor of the feces is, to a large extent, due 
to the presence of indol and skatol and in some cases also to hydrogen 
sulphide, methane, and phosphine. A most disagreeable odor is met 
with in the so-called acholic stools. The odor of fatty acids is observed 
in the lighter grades of infantile diarrhea, while a markedly putrid 
odor is associated with its severer forms. A very characteristic, 
sperm-like odor is noted in the stools of cholera, owing to the pres- 
ence of considerable quantities of cadaverin. A truly rotten stench 
is present in the gangrenous form of dysentery, and in carcinomatous 
and syphilitic ulceration of the rectum. An ammoniacal odor is 
due to an admixture of urine undergoing ammoniacal decomposition. 

Color. — The color of the feces varies, according to the nature of 
the food ingested, from a light to almost a blackish brown, a firm 
stool being in general darker than a thin stool. A stool that has 
remained exposed to the air is also somewhat darker upon its outer 
surface than in its interior, owing to processes of oxidation. In 
nursing infants, in consequence of the exclusive ingestion of milk, 
the color is light yellow. 

Under normal conditions the color is never due to native biliary 
coloring matter, but is largely dependent upon the presence of uro- 
bilin. It is, furthermore, influenced by the nature of the food, 
chlorophyll tending to produce a greenish color, starches a yellowish 
tinge. If much blood is present in the food the feces may be almost 
black, owing to the formation of hematin. Huckleberries and red 
wine likewise produce a blackish color, chocolate and cocoa a gray; 
preparations of iron, manganese, and bismuth color the feces dark 
brown or black, owing to the formation of sulphides of these metals; 
the green color of calomel stools was formerly supposed to be due to 
the formation of a sulphide, but is more likely caused by the presence 
of biliverdin. Santonin, rheum, and senna produce a yellow color. 
Quite characteristic also are the ipecacuanha stools, which closely 
resemble the so-called acholic stools. 

The color of the feces in disease may vary a great deal. When 
unaltered bile is present, the stools may assume a golden-yellow, 
a greenish-yellow, or even a green color. In cases of biliary ob- 
struction or suppression, on the other hand, they become pasty and 
have a grayish or even a white color. This, however, is not so 
much due to the absence of coloring matter derived from the bile as 
to an insufficient absorption of fats, as was shown by Striimpell, who 
succeeded in obtaining stools of a light-brown color after feeding 
patients affected with catarrhal jaundice upon a diet containing 
minimal amounts of fat. Such acholic or colorless stools, as it would 
be better to say, are not only found associated with biliary obstruc- 
tion, but may also occur when the ducts are patent, They have been 



EXAMINATION OF NORMAL FECES 259 

observed in various cases of leukemia, carcinoma of the stomach or in- 
testine, in simple infantile enteritis, chronic nephritis, chlorosis, scarla- 
tina, tuberculous enteritis, and especially frequently in debilitated con- 
sumptives and in cases of chronic tuberculous peritonitis in children. 
In some of these conditions, as in tuberculosis of the intestines and of 
the peritoneum, the lack of color is probably due to a diminished 
absorption of fats. In others, however, this explanation does not 
hold good, as abnormally large amounts of fat are not necessarily 
present. In such cases the lack of color is probably referable to the 
formation of colorless decomposition products of bilirubin, such as 
the leuko-urobilin of Nencki. In this connection it may be interest- 
ing to note that in those cases in which the biliary ducts are patent the 
color of the stools may vary not only from day to day, but even within 
the twenty-four hours. A neurasthenic patient occurring in my prac- 
tice thus passed an acholic stool almost every morning and usually 
colored feces in the afternoon, for a period of several weeks. 

Generally speaking, the color of the stools becomes lighter the 
larger the number of movements, and vice versa. In Asiatic cholera 
and dysentery they may be colorless, while in severe constipation 
the scybalous masses are almost black. 

An admixture of pus in notable amounts also gives rise to a charac- 
teristic color, as is seen in cases of dysentery, syphilitic and carcino- 
matous ulceration of the colon and rectum, following the perforation 
of a parametritic or periproctitic abscess into the rectum, etc. 

Carter and MacMunn 1 have recently pointed out that at times a 
chromogen may be present in the feces, which on exposure to the 
air is transformed into a red pigment, simulating blood-coloring 
matter. They report three cases in which this was observed. Mac- 
Munn expresses the opinion that the substance in question is closely 
related to stercobilin. The stools showed streaks of red upon the 
surface, and after further exposure and repeated agitation turned a 
pronounced blood red throughout. 

Green stools are observed especially in infants, and may be refer- 
able to two different causes, being dependent on the one hand upon 
the presence of a bacillus, described by Le Sage, which produces a 
green coloring matter, while on the other it may be referable to 
biliverdin. When green stools occur frequently, this condition is 
associated with the clinical symptoms of a severe cholera infantum. 
Such stools have also been noted in dysentery referable to infection 
with the Bacillus pyocyaneus. 

If blood is present the stools may present a scarlet red, a dirty 
brownish red, a coffee, or even a perfectly black color. Adherent 
blood, usually bright red in color and found on scybalous masses, is 
probably always derived from the rectum or anus, while a change in 

* Prit. Med. Jour., 1899, 



260 THE FECES 

color, indicating an earlier date of the bleeding, usually points to 
the colon. 

An intimate admixture of blood with the stool, the color being at 
the same time altered, so as to vary from a brownish red to black 
(owing to the presence of ferrous sulphide), is indicative of hemor- 
rhage into the stomach or the small intestine. The darker the color 
the more remote from the anus will be, as a rule, the seat of the 
hemorrhage. Black or coffee-colored stools are thus observed in 
cases of ulcer of the stomach or of the duodenum, in melena neona- 
torum, and similar conditions. 

When profuse intestinal hemorrhages take place, however, as in 
some cases of typhoid fever and melena, and particularly when 
diarrhea exists at the same time, the blood which appears in the 
stools may be changed very little or not at all. 

While simple inspection or a microscopic examination of the feces 
will often determine whether or not blood is present, it has been 
ascertained that occult bleeding may frequently occur where the 
presence of blood can only be established by special chemical exami- 
nation. Evidence of such occult bleeding can be obtained in ma- 
lignant growths involving the gastro-intestinal tract, in ulcus (over 
80 per cent, of the cases), hemorrhagic pancreatitis, catarrhal jaundice 
(at the height of the disease), general venous stasis referable to heart 
lesion. Other sources of bleeding must of course be excluded, and the 
diet during the period of examination should be free from meats. 1 
The aloin test is best employed. 

Aloin Test. — If the stools are not in a semiliquid condition they 
must be made so by thoroughly mixing them with distilled water; 5 
grams of stool are usually sufficient. The material is then extracted 
by shaking with an equal volume of ether. The mixture is allowed 
to stand for fifteen minutes or longer and the supernatant fluid poured 
off. The remaining fecal material is mixed with one-third its volume 
of glacial acetic acid and 10 c.c. of ether. The mixture is again 
thoroughly shaken and set aside for the ethereal layer to separate out, 
and this then poured off. 

The aloin solution which is now used is prepared by dissolving as 
much aloin as will go on the end of a spatula in one-third of a test 
tube of 70 per cent, alcohol; 2 to 3 c.c. of the clear yellow solution 
are mixed in a test-tube with about the same amount of the acetic 
ethereal extract and treated with 2 to 3 c.c. of ozonized turpentine 
(prepared by allowing chemically pure turpentine, such as that of 
Merck, to stand exposed to the air for at least three weeks), or 
an equal amount of active hydrogen peroxide. The mixture 
is thoroughly shaken. If blood is present the reaction may appear 

1 Steele and Butt, Amer. Journ,, Julv, 1905, p. 36. Hartmann, Arch. f. Verdau- 
ungsk., 1904, vol. x, Heft 1. Joachim, Berlin, klin. Woch., 1904, No. 18. 



EXAMINATION OF NORMAL FECES 261 

in one of several ways: either the whole mixture turns pink, which 
gradually deepens to a cherry red, or the solution of aloin sinks 
to the bottom and forms a layer beneath the mixture of ether and 
turpentine, and this lower layer of aloin in positive tests gradually 
becomes a deep cherry red. Sometimes if the ether and turpentine 
are first mixed and the aloin is then allowed to flow gently down the 
side of the tube, the two sets of fluid will remain separate and a deep- 
red ring will form at their junction. Not more than fifteen minutes 
should be allowed for the red color to show itself, for after this the 
aloin will gradually turn red even if blood is not present. It is neces- 
sary to make the aloin solution freshly, for when it stands exposed 
to the light it changes to about the color that it attains in the reaction 
when blood is present. 

If the test is negative the color remains a light yellow, which 
becomes red after standing for some length of time. 

Guaiac Test. — This test may also be employed, but is not quite so 
satisfactory as the one preceding. The ethereal extract of the fecal 
material is prepared as described. The reagent is made by shaking 
a gram or so of gum guaiac in a test-tube half-full of ether and allowing 
the mixture to stand until it becomes clear by settling. A couple of 
c.c. of this solution are added to the same amount of the ethereal 
extract of the feces and at least an equal volume of hydrogen dioxide 
is added. The whole is shaken; the hydrogen dioxide settles to the 
bottom and the ethereal extract floats on top. The blue color (owing 
to the oxidation of the guaiaconic acid to guaiac blue) of a positive 
reaction shows itself very quickly in the supernatant fluid, which in a 
decided reaction becomes a deep blue, that may be somewhat masked 
by the brown color of the urobilin in the ethereal extract. In such a 
case the blue color often becomes a purplish brown, but even this 
reaction is unmistakable. If the reaction is negative no color change 
occurs. The guaiac solution must be fresh, but need not be made 
up daily. 

Macroscopic Constituents. 

Alimentary Detritus. — Upon gross examination of the feces it 
is possible to find stones of cherries, grape seeds, woody vegetable 
fiber, the skins of berries, large pieces of connective tissue, undigested 
pieces of apple, pear, potato, grains of corn, etc. 

The presence of notable amounts of digestible food, such as pieces 
of muscle tissue, flakes of casein, fragments of amylaceous food, con- 
stituting what was formerly spoken of as lientery, is always indicative 
of disturbed gastric or intestinal digestion. It is hence observed in 
chronic intestinal catarrh, febrile dyspepsia, etc. Occasionally also 
unaltered food in large amounts is found in the feces, owing to a. 
direct communication between the stomach and the colon, as in cases 
of perforating ulcer or carcinoma of the stomach. 



262 THE FECES 

When fat is present in abnormally large amounts it can usually be 
recognized with the naked eye. To this condition the term steatorrhea 
has been applied. In typical cases the fat is seen in the form of 
whitish or grayish masses, varying in size from that of a pea to that 
of a walnut, which are more or less intimately mixed with the fecal 
material, and may at first sight be mistaken for flakes of casein. 
From these it may be distinguished by its chemical reactions and its 
peculiarly glistening appearance. In other cases stools may be seen 
in which the fecal column is covered, to a greater or less extent, with 
a grayish, dense, asbestos-like substance, while the core itself presents 
the usual color. Nothnagel states that this appearance is referable 
to congealment of the fat when it is exposed to a lower temperature 
than that of the body. I have repeatedly observed this appearance 
in stools which had just been voided and were still warm. In other 
cases the fat is intimately mixed with the feces, which are colored a 
light gray throughout. The passage of liquid oil in the absence of 
fecal material has also been recorded, but it seems doubtful that the 
oil in such cases entered the body by the mouth. Following the use of 
oil enemas such stools are, of course, seen. 

The elimination of abnormally large quantities of fat may be due 
to the ingestion of correspondingly large amounts. More frequently, 
however, it is referable to pathological conditions. A steatorrhea 
will thus naturally occur when an insufficient supply of bile is poured 
into the small intestine, and hence is observed constantly in cases 
of biliary obstruction. True steatorrhea is also met with in dis- 
eases affecting the resorptive power of the small intestine, such as 
extensive atrophy or amyloid degeneration of the intestinal mucosa, 
tuberculous ulceration, etc., or in diseases involving the integrity of 
the lymphatic glands and vessels of the mesentery, as in chronic 
tuberculous peritonitis, caseous degeneration of the mesenteric glands, 
etc. In simple catarrhal conditions, however, steatorrhea may also 
occur, and not only in infants, but, according to my experience, also 
in adults. The question whether or not steatorrhea is constantly 
observed in cases of pancreatic disease, as some observers have 
claimed, may now be answered in the negative, although it must be 
admitted that the two conditions are very frequently associated. Le 
Nobel, who has investigated this subject, arrived at the conclusion 
that the steatorrhea in itself is of little practical importance, but that 
its association with the absence of products of putrefaction from the 
stools, the absence of the salts of the fatty acids, and the presence of 
maltose in the urine, may possibly be regarded as indicating the 
existence of pancreatic disease. 

Mucus and Mucous Cylinders.— So long as mucus occurs in 
small particles only, adherent to otherwise normal feces, it is of no 
pathological significance. Larger amounts are almost always indic- 
ative of a catarrhal condition of the colon or rectum, no matter 



EXAMINATION OF NORMAL FECES 263 

whether the stool is otherwise normal or whether diarrhea exists at 
the time. Peculiar formations are occasionally seen, viz., so-called 
mucous cylinders, which are passed in large or small fragments in a 
condition which has been described by Nothnagel as enteritis mem- 
branosa or colica mucosa. 1 Such masses, which at times measure a 
foot or more in length, are ribbon or net shaped, and are frequently 
passed in the absence of fecal matter, with severe tenesmus. They 
resemble Curschmann's spirals, but lack the central thread and the 
Charcot-Leyden crystals. They are probably indicative of chronic 
constipation associated with catarrh of the colon. Not to be con- 
founded with this condition is the passage of masses of mucus, which 
do not present the cylindrical form, but which also may be passed 
with a great deal of tenesmus and in the absence of fecal matter; 
this is very commonly seen in cases of nephroptosis associated with 
gastroptosis and enteroptosis. These formations are in all probability 
also referable to a catarrhal condition of the colon. In cholera 
Asiatica particles of mucus are seen which resemble grains of rice; 
their presence was formerly regarded as characteristic of this disease, 
but they are now known to occur in ordinary catarrhal conditions 
also. 

Biliary and Intestinal Concretions. — Most important from a 
diagnostic standpoint is the examination of the feces for the presence 
of biliary concretions, which should never be neglected in cases of 
colicky, abdominal pain of doubtful origin, whether associated with 
jaundice or not. 

When searching for gallstones the feces should be stirred with 
water and passed through a fine sieve. Biliary concretions may then 
be found as small, crumbling masses, or as hard stones presenting an 
irregular contour or the smooth, characteristic facets. In size they 
may vary from that of a millet seed to that of a pigeon's egg; large 
stones are rarely passed by the bowel unless perforation has occurred 
into the intestines and usually into the colon. 

Some calculi consist almost entirely of cholesterin, while others 
are composed essentially of inspissated bile, and still others of cal- 
careous salts. The former are the most common, and are readily 
recognized by their softness and color, which may be white, grayish, 
bluish, or greenish. Their specific gravity is lower than that of 
water. Very frequently they contain a nucleus, composed of earthy 
sulphates or phosphates. An analysis which I made of a stone of 
this kind, weighing 10.548 grams, gave the following results: 

1 Nothnagel, "Colica Mucosa," Beitrage z. Physiol, u. Path. d. Darmes, 1884. 
Fleiner, Berlin, klin. Woch., 1893, Nos. 3 and 4. Einhorn, Arch. f. Verdauungs- 
krank., vol. iv, p. 456 



264 THE FECES 

Cholesterin 72 . 590 per cent 

Mineral salts 0.247 " 

Fats 5.090 

Biliary pigments 13.930 " 

Organic matter 7.270 " 

Calculi which consist largely of biliary pigments are brown in color. 
They are hard, and heavier than water. Frequently they contain 
traces of copper and zinc (Fig. 71). 

Calculi composed of calcareous salts generally present an irregular, 
roughened contour. 

Welch has drawn attention to the not infrequent presence of pure 
colonies of the Bacillus coli communis in gallstones, apparently 
forming their nucleus. Typhoid bacilli also have since been observed 
in their interior, and it appears likely that the formation of gall- 
stones is primarily referable to an invasion of the gall-bladder by such 
microorganisms. A remarkable case has been reported by Pearce, 




Fig. 71. — Gallstones: a, cholesterin; b, pigment stones. 

in which a leptothrix was the only microorganism found in biliary 
concretions, while in the bile this was present together with the colon 
bacillus. 1 

Intestinal concretions (enteroliths) are rare and usually come from 
the appendix. At times they contain some foreign body, such as a 
grape seed, as a nucleus, upon which calcium and magnesium salts 
have become deposited. 

Fecal calculi or coproliths are likewise only rarely seen. They 
represent inspissated fecal material which has become impregnated 
with lime and magnesium salts. More commonly they are found at 
the postmortem table in the cecum, in the haustra of the colon, and 
in the rectum. 

Intestinal sand is also rare. I have seen only 5 cases in the past 
ten years. In the German literature I have found reports of only 3 
cases, while in the French literature about 16 have been recorded. 
Of its origin nothing is known. The condition is commonly associated 
with enteritis membranacea. The material presents a brownish color, 

1 Pearce, Univ. of Penna. Bull., Aug., 1901. Gushing "On the Presence of 
Typhoid Bacillus," Johns Hopkins Hospital Bull., 1899, p. 166; and Hunner, 
ibid., 1898, p. 163. Cushing, "On the Presence of the Colon Bacillus," ibid.; 
and Fournier, cit. by Chauffard, Rev. d. med., 1897, p. 81. 



MICROSCOPIC EXAMINATION OF THE FECES 265 

but may be light green.. In 1 case reported by Deetz 1 it was pos- 
sible to demonstrate the presence of calcium phosphate with traces of 
calcium oxalate. One case is recorded by Thomson and Ferguson. 2 
Analysis: 11.7 per cent, of CaCO s ; 87.3 per cent, of Ca 3 (P0 4 ) 2 ; 
insoluble residue (silica), 1 per cent. There was present also a pig- 
ment which the writers regard as intermediary between ordinary bile 
pigment and stercobilin. They think the sand is formed in the ileum. 
Foreign Bodies. — In children, the insane, in cases of hysteria, 
and even in people who are apparently possessed of their normal 
senses, the physician must be prepared to find at times all kinds of 
foreign bodies, such as pins, coins, buttons, false teeth, tooth-plates 
with ragged edges, and even dirk-knives, all of which have been 
known to pass through the alimentary canal. It must not be for- 
gotten, however, that in cases of hysteria bodies may be shown by 
patients which they claim have passed by the rectum, but which 
have been wilfully added to the stools, such as snakes, frogs, etc. 



MICROSCOPIC EXAMINATION OF THE FECES. 
General Technique. 

The general technique in the microscopic examination of the 
feces is very simple. Stools that are firm when passed should be 
stirred up with water to a moderately thin mush. Drops of this 
material are mounted on a series of slides, covered with cover-glasses, 
and examined at first with a low power (f B. & L.) and then with a 
medium power (-g- or \). The survey with the low power furnishes 
a general idea of the amount of food remnants (muscle fibers, frag- 
ments of vegetable material, fat), of the presence of crystals, pus, blood, 
and eggs of parasites. The higher power (J or y) is reserved for gen- 
eral purposes of verification, to make out details of structure, and the 
search for the smaller animal parasites (trichomonads, ameba coli, etc.) 

If the stools are already thin when passed, no further dilution is 
necessary. Bits of mucopus or of material showing the presence 
of blood are generally advantageous for the search for amebas. 
Musgrave and Clegg, however, recommend that in doubtful cases 
it is well to administer a saline cathartic and to examine the fluid 
portion of the resulting movements. In the examination for amebas it 

1 Deutsch. Arch. f. klin. Med., 1901, vol. lxx, p. 365. See also Dieulafoy, "La 
lithiase intestinale et la gravelle de l'intestin," Presse med., March 10, 1897 
(extract in Centralbl. f. klin. Med., 1897, p. 904). 

2 Jour, of Pathol, and Bad., vol. vi, 1900, p. 334. Laboulbene, Bull. Acad, de 
med., Paris, 1873. Sheridan, Trans. Path. Soc. London, 1890, vol. xli, p. 111. 
D. Thoma, Australasian Med. Gaz., Nov., 1891. Mathieu and Richaud, Soc. 
med. d. hop., May 22, 1896. S. J. Shattock, Trans. Path. Soc. London, vol. 
xlviii. 



266 THE FECES 

is essential that the stools be passed into a warmed bedpan and 
examined at once on warmed slides or by the aid of a warm stage. 
A convenient form of warm stage, which may be obtained from instru- 
ment-makers at low cost, is composed of brass and made to be held 
in position on the stage of the microscope by spring clips. It is about 
8 cm. long and 3 cm. broad, and has cemented to a recessed bottom 
an ordinary glass slip; an opening measuring 1.35 cm. in diameter is 
in the centre of the stage. To one of the long slides of the brass stage 
is fitted a projecting stem, about 10 cm. long, to which the heat of a 
spirit-lamp is applied. 

Specimens containing eggs of parasites are readily preserved by 
the addition of 5 per cent, carbolic acid or of thymol. 







r - . ; " 
















-.-•-> ■ a 


'V'^^-''^ ^ 











Fig. 72. — Collective view of the feces: a, muscle fibers; b, starch granules; c, vegetable 
material; d, potato cells; e, egg of Uncinaria duodenalis; /, calcium oxalate crystals; g, 
fatty acid crystals; h, Charcot-Leyden crystals. 

o 

Unless living organisms are to be searched for, the stools if liquid 
may be placed in conical glasses and covered with a layer of ether so 
as to diminish the disagreeable odor; if mushy or firm they may be 
spread upon a plate and covered with a layer of turpentine. 

Constituents Derived from Food. — Microscopically, indigestible 
and undigested constituents of food may be seen (Fig. 72), such as 
the framework of vegetable material, sometimes still containing 
starch granules or remnants of chlorophyll; muscle fibers, usually 
colored yellow and more or less altered in structure. Elastic-tissue 
fibers are readily recognized by their double contour and bold out- 
lines. Connective-tissue fibers of the white variety can also generally 
be distinguished; when present in large quantities they are usually 



MICROSCOPIC EXAMINATION OF THE FECES 267 

indicative of some digestive derangement, unless they are observed 
following the ingestion of a meal particularly rich in meat. Flakes 
of casein also are seen frequently. 

Muscle fibers are found in every stool whenever meat has been 
eaten. Under normal conditions, however, they are not numerous, 
unless particulary large quantities have been ingested. Their ap- 
pearance under the microscope may vary considerably. On the 
one hand, fibers are met with which still retain their characteristic 
features; others are split up either partially or entirely into the well- 
known disks; but more common than both are more or less roundish, 
yellow, apparently homogeneous fragments, which at first sight do not 
resemble muscle fibers in the least. Upon closer investigation, how- 
ever, their true nature will become apparent. It will then be seen 
that two of the sides in some portions at least are more or less par- 
allel, and if the specimen is examined with a high-power lens some 
traces of cross-striation can probably always be discovered. 

Isolated starch granules are scarcely ever found under normal 
conditions, excepting in young children who have been fed with much 
starchy material. Starch granules enclosed in vegetable cells are 
likewise not found as a general rule, but are more common than the 
isolated granules. Their presence is easily recognized by treating 
microscopic preparations with a solution of iodopotassic iodide 
(Lugol's solution), when the granules or fragments will assume a blue 
color. 

The presence of fat in the feces is quite constant, even in health. 
It may occur in the form of needle-like crystals, as fat droplets, or 
as polygonal masses which are highly refractive and often colored 
yellow or a yellowish red. Their true nature is easily recognized 
by adding a drop of concentrated sulphuric acid and heating, when 
they are transformed into the characteristic fat droplets. 

The so-called acholic stools are usually very rich in fat, and particu- 
larly so in cases of biliary obstruction associated with jaundice. At 
other times the lack of color, as has been mentioned above, is not 
referable to the secretion of an insufficient amount of bile, but to 
the presence of colorless decomposition products of bilirubin, such 
as the leuko-urobilin of Nencki. In these cases abnormally large 
quantities of fat are not always present. The conclusion that a stool 
contains excessive amounts of fat because it is apparently acholic is 
hence not justifiable unless a microscopic examination has been 
made. 

In pathological conditions it is necessary to determine whether or 
not food remnants are present in abnormal amount, presupposing, of 
course, that excessive quantities have not been ingested. It is often 
possible to draw definite conclusions as to the state of intestinal 
digestion from the excess of one form of non-digested material over 
another. The presence of large quantities of undigested starch 



268 



THE FECES 




indicates a catarrhal condition of the small intestine, and it may, 
indeed, be said that the occurrence of more than traces of this mate- 
rial should be regarded with suspicion. An increase in the number 
of muscle fibers will, as a rule, likewise be observed under such 
conditions. 1 

Schmidt and Strassburger 2 have described a special form of intes- 
tinal fermentative dyspepsia, in which there is an isolated amylo- 
lytic insufficiency, which may be of functional or of organic origin. 
(See Schmidt's fermentation test below.) 

In this connection it is noteworthy that in man extensive disease 
of the pancreas may exist without seriously disturbing amylolytic 
digestion. 

Schmidt's Fermentation Test. — To obtain a more exact insight 
into the degree of amylolytic insufficiency of the intestinal tract than 
is possible from a microscopic study of the feces, 
Schmidt has proposed a special method which 
is based upon the continued digestion of the 
carbohydrates in the feces. The examination 
is made after the patient has been placed on the 
following test diet (Schmidt and Strassburger's 
test diet No. II) : milk, 1.5 liters; 3 J eggs; strained 
oatmeal gruel (from 80 grams of oatmeal); 100 
grams of Zwieback; 20 grams of butter; 20 grams 
„ e of sugar; 125 grams of steak (raw weight), and 190 
grams of potato (raw weight) . The distribu- 
tion of these various articles of food can be 
arranged as one chooses, or as follows: At 7.30 
a.m., f liter of milk and 2 Zwiebacks (each 33 
grams); at 10.30 a.m., | liter of bouillon with | 
egg; at 12 m. f liter of milk with 1 egg; between 
1 and 2 p.m. \ liter of oatmeal gruel (prepared 
from 40 grams of oatmeal, 166 grams of milk, 
10 grams of sugar, and \ egg); 100 grams of 
well-done Hamburg steak (125 grams of raw 
beef, raw weight) and 12 grams of butter; 250 
grams of mashed potato (from 190 grams of 
potato, 60 grams of milk, and 8 grams of butter); 
at 4.30 p.m., f liter of milk, 1 egg, 1 Zwieback; 
at 7.30 p.m., \ liter of oatmeal gruel as at dinner- 
time. Before commencing with the test diet, how- 
ever, it is necessary to demarcate the fecal material 
by giving a wafer or capsule containing 0.3 gram of powdered car- 
mine. The examination proper is made as soon as the feces are no 

1 Schmidt u. Strassburger, Deutsch. Arch. f. klin. Med., vol. lxix, p. 570. 

2 "Die klinische Bedeutung der Ausscheidung von Fleischresten mit dem 
Stuhlgang," Deutsch. med. Woch., 1899, p. 811. 




Fig. 73.— Schmidt's fer- 
mentation tubee. 



MICROSCOPIC EXAMINATION OF THE FECES 269 

longer colored red, viz., after from two to three days of the test diet. 
The necessary apparatus is pictured in the accompanying figure 
(Fig. 73), which represents one-third of the acutal size. For each 
experiment 5 grams of fresh fecal material are used (the feces being 
of medium consistence; otherwise a little more or less is taken, corre- 
sponding to about 1 gram of dry residue). The material is well 
stirred with water in the bottle a, which is filled entirely and closed 
with the rubber stopper, care being taken to exclude bubbles of air. 
Tube b is filled with water from the tap and also closed without 
admission of air. Tube c should contain no water; it has a pinhole 
aperture at the top. The communicating tube d is adjusted as 
shown in the figure. The apparatus is then placed in the incubator 
at 37° C. for twenty-four hours, not longer. During this time the 
carbohydrate fermentation will have been completed (Schmidt's 
Fruhgahrung) . During the evolution of gas water will be dis- 
placed from b into c; the resulting column is measured and repre- 
sents the degree of fermentation. The result is regarded as positive 
if more than a quarter tubeful of gas is obtained. With the test 
diet in question this would mean a condition approximating the 
normal. In such an event the patient is placed for two days fur- 
ther on test diet No. I, which differs from No. II only in the absence 
of the meat and potato. If then there is still a positive result, the 
diagnosis of "fermentative dyspepsia" is justifiable. In order to 
eliminate errors arising from possible formation of gas as the result 
of albuminous putrefaction the fermenting fecal material should be 
tested from time to time in a control specimen. If the formation of 
gas is due to carbohydrate fermentation, there will be an increasing 
degree of acidity (tested with litmus paper); this increase, however, 
is not always marked; at any rate, there must be no increasing 
alkalinity. 

Leiner's Test for Casein. — Casein is most conveniently demon- 
strated with Leiner's method. To this end a small amount of 
fecal matter is spread on a slide and dried in the air. It is then fixed 
by heat — passing the specimen through the flame of a Bunsen burner 
three or four times is sufficient — and stained with a mixture of equal 
parts of a 0.75 per cent, solution of acid fuchsin and methyl green in 
50 per cent, alcohol, the mixture being diluted ten times with water. 
After fifteen minutes the preparations are placed in distilled water 
and allowed to remain for one hour or longer. Casein and para- 
casein are thus stained a pale blue or violet, while similar bodies are 
practically all colored a light green, or more rarely a yellowish 
green. 

Determination of the Residual Albumin (Koziczkowsky). — The 
patient is placed upon a test diet very similar to that of Schmidt and 
Strassburger, consisting of lj liters of milk, \ liter of bouillon, 6 pieces 
of Zwieback, 40 grams of oatmeal, 40 grams of butter, 2 eggs, 30 grams 



270 THE FECES 

of finely hashed meat, and 200 grams of potato. The feces are pre- 
viously demarcated by giving 0.3 gram of powdered carmine. 

Two portions of stool, each representing 2 grams of dried feces, 1 
are placed upon nitrogen-free filters and washed successively with 
ordinary ethyl alcohol (93 to 94 per cent.), absolute alcohol, and 3 per 
cent, hydrochloric acid. One portion (A) is then mixed with 50 c.c. 
of a digestive mixture of the following composition: 

3 per cent, solution of hydrochloric acid 10.0 

Pepsin 30.0 

Water 100.0 

The second portion (B) is suspended in a corresponding amount 
of dilute hydrochloric acid without pepsin. The total acidity and 
amount of free hydrochloric acid are then estimated in each by titrating 
with T ^- alkali solution, after which both specimens are corked and 
placed in the incubator over night, at 37° C. The next day the total 
acidity and free acid are again estimated. The difference in the 
amount of free acid in specimen A indicates the amount which was 
used in the digestion of the albumins present, and thus serves as an 
index of their quantity; normally this corresponds to from 15 to 18 
c.c. of y-Q normal alkali. The difference in the amount of free acid in 
specimen B is referable to the action of proteolytic ferments (pepsin) 
in the feces per se. Normally this rarely exceeds 2 to 3 c.c. y-g normal 
solution. 

Morphological Elements Derived from the Alimentary Canal. 
Epithelium. — Well-preserved cylindrical or goblet cells are only 
exceptionally found in the feces, while transition forms from the nor- 
mal cells to mere spindles, in which a nucleus can no longer be recog- 
nized, are observed quite constantly. These degenerative changes, 
according to Nothnagel, 2 are the result of an abstraction of water 
from the cells, which may alter their appearance to an extent that only 
the experienced eye is capable of recognizing their true character. 
Pavement epithelial cells, when present, are derived from the anal 
orifice. 

Epithelial cells when present in large numbers always indicate an 
inflammatory condition of some portion of the intestinal tract. 

Cylindrical epithelial cells are found in abundance in all inflam- 
matory conditions affecting the intestinal mucosa. They are almost 
exclusively seen embedded in mucus, and it is interesting to note that 
the cloudy appearance of the mucus is referable to the presence of 
these elements, and not to leukocytes, as is the case in the sputum. 

1 1 gram of formed stool represents 0.3 gram, of the dried substance; 1 gram of 
semiliquid stool (good fat absorption) equals about 0.25 to 0.27 gram; 1 gram of 
semiliquid stool (with poor fat absorption) equals about 0.116 gram of dry 
material. 

2 Beitrage z. Physiol, u. Pathol, d. Darmes, Hirschwald, Berlin, 1884, and 
Spezielle Pathol, u. Therap., Holder, Wien, 1895, vol. xvii, pt. i. 



MICROSCOPIC EXAMINATION OF THE FECES 



271 



When bile-stained specimens are met with, the conclusion is justifi- 
able that the small intestine is involved. 

Epithelioid cells may be found in carcinoma of the rectum. 

Leukocytes. — Leukocytes are almost always absent in normal 
stools or present only in very small numbers. Large numbers usually 
indicate a severe catarrhal, if not an ulcerative, condition of the intes- 
tines. Pure pus in large amounts is observed especially in dysentery 
and in cases in which abscesses have perforated into the gut from 
adjacent organs or cavities. 

Red Blood Corpuscles.— Unaltered red blood corpuscles, accord- 
ing to Nothnagel, are but rarely observed in the feces, no matter 
how intensely red they may be colored, providing that an ulcerative 
process affecting the colon or the rectum can be excluded; in that 
case, as in the severer forms of dysentery, large numbers may be 
observed. If the hemorrhage has occurred higher up in the intes- 
tine, large and small masses of a brownish-red color are seen, which 
consist of hematoidin. They are mostly amorphous, but in some 
specimens the characteristic rhombic crystals may be observed. In 
general, it may be said that the higher the seat of the hemorrhage 
the darker will be the color of the pigment, and the less the chances 
of finding well-defined red corpuscles. In such cases recourse must 
be had to the tests for occult blood (which see). 




rIv/;:--.v.'.:.: 



Fig. 74. — Fatty crystals obtained from the feces. 



Crystals. — Needle-like crystals of free fatty acids, and the cal- 
cium and magnesium salts of the higher members of this group, 
occurring either singly or arranged in sheaves, may be found in 
every stool (Fig. 74). They are of no significance unless present 
in Jarge numbers. Nothnagel speaks of the frequent occurrence 
of certain calcium salts (of fatty acids, as he believes) in normal as 
well as pathological stools. He states that they are almost always 



272 THE FECES 

bile-stained, and occur in irregular, sometimes elliptical, oval, or cir- 
cular masses, in which a crystalline structure cannot be distinguished. 
They are apparently of no importance. Quite common, also, are 
crystals of neutral calcium phosphate and ammoniomagnesium phos- 
phate, the former occurring in the form of more or less well-defined, 
wedge-shaped crystals collected into rosettes, the latter presenting 
the well-known coffin-shape when the stool is mushy, while in firm 
stools irregular fragments mostly are found. At one time the ammo- 
nio-magnesium phosphate crystals were supposed to be characteristic 
of typhoid stools, but it is now known that they occur in normal feces, 
as well as under the most varied pathological conditions. Their 
presence is hence of no diagnostic significance. It is important to 
note that the neutral phosphates are never stained by bile pigment, 
and the triple phosphates only in rare instances. Both are easily 
soluble in acetic acid. Crystals of calcium oxalate may be found in 
abundance following the ingestion of certain vegetables, such as 



V 



K A Hs 



&■ 



W^gWS 



Fig. 75. — Cholesterin crystals. 

sorrel and spinach. They are usually found embedded in the vege- 
table debris. They are readily recognized by their characteristic 
envelope form, their insolubility in acetic acid, and their solubility 
in hydrochloric acid. Not infrequently they are bile-stained. 

Calcium lactate is frequently seen in the stools of children receiving 
a milk diet; it occurs in the form of sheaves composed of radiating 
needles. Calcium carbonate is rarely observed, but occasionally 
occurs in the form of amorphous granules or dumb-bell-shaped crys- 
tals. Calcium sulphate crystals are likewise rare, but may be pro- 
duced artificially by the addition of sulphuric acid, when beautiful 
needles and platelets may be observed. Cholesterin, while always 
present in solution, is rarely observed in crystalline form (Fig. 75). 
Hematoidin crystals are never found in normal stools. Charcot- 
Leyden crystals, according to my experience, are not found in normal 
stools. They have been described in cases of typhoid fever, dysentery, 
and phthisis, but are rare in these diseases. In uncinariasis they are 



BACTERIOLOGY OF THE FECES 273 

more frequently seen, but not in every case. Often they only form 
after the stool has been kept for some time. They are more likely 
to be encountered when there are many eggs present than in milder 
cases. They have further been seen in association with Ascaris lum- 
bricoides, Oxyuris vermicularis, Taenia solium and saginata. In cases 
of trichocephalus they are but rarely seen, while they are always 
absent in the case of Taenia nana. According to Leichtenstern 1 their 
persistence in the feces after the evacuation of what would appear to 
be a complete taenia should be regarded as indicating the non-removal 
of the head. In amebic colitis these crystals have also been observed 
by Lewis, Lafleur, Amberg, myself, and others. 

Mucus. — Small hyaline particles of mucus, visible only with the 
microscope, are not infrequently met with under pathological condi- 
tions, and are of diagnostic significance. When bile-stained, their 
presence is always indicative of disease of the small intestine proper, 
while colorless particles point to a catarrhal condition of the upper 
portion of the large intestine or the lower portion of the small intes- 
tine. Beginners should be careful not to mistake apparently hyaline 
particles of vegetable residue for mucus. Mucus never yields a 
blue color when treated with iodine, or iodine and sulphuric acid, 
and examination with a higher power will show the entire absence 
of any definite structure. Both forms, viz., colorless and colored 
particles, are found intimately mixed with the feces, and may be very 
abundant. In addition to these forms Nothnagel has described the 
occasional occurrence of large numbers of roundish or irregular, 
very pale hyaline or opaque formations, which are devoid of all 
structure. Some specimens are homogeneous, while others present a 
distinct rimous appearance. They have been found only in liquid 
stools, and are apparently of no diagnostic significance. To judge 
from their optic behavior, they probably consist of mucus. 2 



BACTERIOLOGY OF THE FECES. 

The bacteria are the microorganisms xar i£o%yju which are found 
in the feces. Their number is truly enormous. SucksdorfT found in 
his own person that on an average 53,124,000,000 were eliminated in 
the twenty-four hours under normal conditions. If we recall the 
strongly bactericidal power of the gastric juice, such an observation 
must at first sight appear surprising. It should be remembered, how- 
ever, that large amounts of the ingesta are carried into the small intes- 
tine at a time when hydrochloric acid has not yet appeared in 
the free state. 

1 Deutsch. med. Woch., 1885, vol. xi, Nos. 29 and 30; ibid., 1886, vol. xii, Nos. 
11 to 14; ibid., 1887, pp. 565, 594, 620, 645, 669, 691, and 712. 

2 A. Schmidt, "Ueber Schleim im Stuhlgang," Zeit. f. klin. Med., vol. xxxii ; 
p. 260. 

18 



274 THE FECES 

On the whole, the bacteriological flora of the intestinal contents 
is fairly constant, but, as in the other cavities and channels of the 
body where bacteria are invariably met with, transient guests are 
also not uncommon. The majority of the bacteria which are here 
encountered are, as a general rule, harmless; but it is important to 
note that under suitable conditions a number of these may develop 
pathogenic properties. Broadly speaking, the bacteria which may 
be found in the feces can be divided into two classes, viz., into alkali 
producers and acid producers. Many of these forms have been de- 
scribed for the first time by Ford, 1 and the following schema, which 
gives a very good idea of the numerous individual types, although 
not complete, is taken from his excellent work: 

Alkali Producers. 

Group I. Organisms producing alkali in litmus milk; not liquefy- 
ing any media; not fermenting carbohydrates to the point of acidity. 
Fecalis alkaligenes, or Petruschky group. Represented by: 
Bacillus alkaligenes. 

Group II. Organisms producing alkali; not liquefying any media; 
fermenting carbohydrates to the point of acidity, but no gas. Dys- 
entericus, or Shiga group. Represented by: 
Bacillus dysenteric. 
Bacillus pseudodysentericus, Muller. 
Bacillus typhi. 
Bacillus acidophilus. 
Group III. Organisms producing alkali; not liquefying any media; 
fermenting the carbohydrates with the production of acidity and gas. 
Hog cholera, or suipestifer group. Represented by: 

Bacillus alkalescens, Ford; ferments dextrose, saccharose, and 

lactose. 
Bacillus subalkalescens, Ford; ferments dextrose, saccharose, and 

lactose. 
Bacillus enteritidis, Gartner; ferments dextrose. 
Bacillus galactophilus, Ford; ferments saccharose and lactose. 
Group IV. Organisms producing alkali; liquefying gelatin; fer- 
menting carbohydrates with the production of acid and gas. Enteri- 
cus group. Represented by: 

Bacillus entericus, Ford; ferments dextrose, saccharose, and 

lactose. 
Bacillus subentericus, Ford; ferments dextrose and lactose. 
Group V. Organisms producing alkali; liquefying gelatin, casein, 
and blood serum; fermenting carbohydrates with the production of 
acid and gas. Proteus, or Hauser group. Represented by: 

1 Studies from the Royal Victoria Hospital, Montreal, vol. i, No. 5, and from 
the Rockefeller Institute for Medical Research, vol. ii. 



BACTERIOLOGY OF THE FECES 275 

Bacillus plebeius, Ford; ferments dextrose, saccharose, and lac- 
tose. 
Bacillus infrequens, Ford; ferments dextrose and lactose. 
Bacillus vulgaris, Hauser; ferments dextrose and saccharose. 
Group VI. Organisms producing alkali; liquefying various media, 
but not fermenting carbohydrates to the point of acidity. Booker 
group. Represented by: 

Bacillus recti, Ford; liquefies gelatin. 

Bacillus pylori, Ford; liquefies gelatin and casein. 

Bacillus ceci, Ford; liquefies gelatin, casein, and blood serum. 

Bacillus Bookeri, Ford; liquefies gelatin, casein, and blood 

serum. 
Bacillus pyocyaneus. 

Acid Producers. 

Group I. Organisms acidifying and coagulating milk; not liquefy- 
ing any media; not fermenting carbohydrates to the point of acidity. 
Fecalis oxy genes, or Bienstock group. Represented by: 
Bacterium oxygenes, Ford. 
Bacterium Bienstock, Schroter. 
Group II. Organisms acidifying and coagulating milk; not lique- 
fying any media; fermenting carbohydrates to the point of acidity, 
but no gas. Acidoformans, or Sternberg group. Represented by: 
Bacillus oxyphilus, Ford. 
Bacterium acidoformans, Sternberg. 
Bacterium minutissimum, Migula. 
Group III. Organisms acidifying and coagulating milk; not 
liquefying any media; fermenting carbohydrates with the production 
of acidity and gas. Coli, or Escherich group. Represented by: 
Bacillus coli, Migula; ferments dextrose and lactose. 
Bacillus communior, Ford; ferments dextrose, saccharose, and 

lactose. 
Bacterium aerogenes, Migula; ferments dextrose, saccharose, and 

lactose. 
Bacterium duodenale, Ford; ferments dextrose and lactose. 
Group IV. Organisms acidifying and coagulating milk; liquefying 
gelatin and fermenting the carbohydrates with the production of 
acidity and gas. Liquefaciens, or Eisenberg group. Represented by: 
Bacillus gastricus, Ford; ferments dextrose, saccharose, and lac- 
tose. 
Bacillus subgastricus, Ford; ferments dextrose and lactose. 
Bacterium liquefaciens, Eisenberg; ferments dextrose, saccharose, 

and lactose. 
Bacterium subliquefaciens, Ford; ferments dextrose and lac- 
tose. 



276 THE FECES 

Group V. Organisms acidifying and coagulating milk; liquefying 
gelatin, casein, and blood serum, and fermenting the carbohydrates 
with the production of acidity and gas. Cloacce, or Jordan group. 
Represented by: 

Bacillus cloacae, Jordan; ferments dextrose, saccharose, and lac- 
tose. 

Bacillus subcloacse, Ford; ferments dextrose and lactose. 

Bacillus iliacus, Ford; ferments dextrose and saccharose. 
Group VI. Organisms acidifying and coagulating milk; liquefying 
various media; fermenting the carbohydrates with the production of 
acidity, but no gas. Dubius, or Kruse group. Represented by: 

Bacillus chylogenes, Ford; liquefies gelatin. 

Bacterium chymogenes, Ford; liquefies gelatin. 

Bacillus leporis, Migula; liquefies gelatin and blood serum. 

Bacillus dubius, Kruse; liquefies gelatin, blood serum, and casein. 

Bacillus jejunalis; liquefies gelatin, blood serum, and casein. 
All the above are non-pigment, non-spore bearing organisms. In 
addition to these the following pigment-producing and spore-bearing 
organisms have been isolated: 

Pseudomonas aeruginosa, Schroter. 

Pseudomonas ovalis, Ravenel. 

Bacterium Havaniense, Sternberg. 

Bacterium lutescens, Migula. 

Bacterium anthracoides, Huppe and Wood. 

Bacterium implectans, Burchard. 

Bacillus cereus, Frankland. 

Bacillus mycoides, Fliigge. 
The above list indicates the various organisms which have thus far 
been isolated from the intestinal contents. Many other forms exist, 
but have not yet been cultivated, as they do not grow on the artificial 
media which are now in use. 
The more important members of the series are described below. 
Fungi. — Fungi, with the exception, perhaps, of the Oidium albi- 
cans, which has at times been observed, are rarely found in the feces. 
Schizomycetes. — Saccharomyces cerevisise is one of the normal 
constituents of the feces, and is found in its characteristic forms, 
three or four buds, however, being but ordinarily observed. Owing 
to the glycogen present in their substance, they assume a mahogany 
color when treated with a solution of iodopotassic iodide. They 
should not be confounded with a class of bacteria which closely re- 
semble the saccharomyces in general appearance, but are colored blue 
when treated in the same manner. 

Bacillus dysenterise, Shiga. — This organism is now generally re- 
garded as the specific cause of the common form of acute dysentery 
which prevails not only in the tropics, but also in the United States 



BACTERIOLOGY OF THE FECES 277 

and Europe. It was discovered by Shiga in Japan in 1897, and 
is identical with the organism obtained by Flexner and Strong in the 
Philippines and Porto Rico, by Vedder and Duval in the United 
States, and by Kruse in Germany. From the researches of Bassett 
and Duval it further appears that the same bacillus is also responsi- 
ble for the common form of infantile summer diarrhea which prevails 
in warm countries. 

In the United States the Flexner-Harris type is by far the most 
common in infantile cases. In the collection of 237 cases reported 
by Holt this type was found in 207, while the true Shiga bacillus 
was present in only 23; both organisms were found in 7 cases. 

The bacillus in question is a short rod with rounded ends, and 
resembles the typhoid bacillus and most members of the colon group. 
It is probably non-motile so far as active locomotion is concerned, 
but it is possessed of a high degree of molecular motion. It stains 
with the usual basic dyes and is decolorized by Gram's method. 

Upon gelatin plates at room temperature there appear, after a few 
days, small round dots, which, magnified under low powers, are 
slightly yellow and finely granular. After a few days they increase 
in size; the middle portion of the colonies then appears darker under 
a low power, while the outer zone appears brighter and more seed-like. 
The superficial and deeper colonies show no marked variation. In 
stab cultures on gelatin a whitish strand forms the whole length of 
the stab. The gelatin is not liquefied. 

After twenty-four hours in the incubator single colonies upon 
slanted agar appear moist, bluish, and partially translucent. After 
two days they present a combination of a middle dark and a periph- 
eral bright, sharply defined zone. 

The growth on glycerin agar is slightly more abundant than on 
ordinary agar. The organism grows on blood serum without lique- 
fying it. 

In the stab cultures on glucose agar there is formed along the 
whole line of the puncture a thick, gray-white strand without the 
development of gas. Upon potato after twenty-four hours in the 
incubator there is hardly any perceptible growth, only the surface 
appears slightly shiny. After two days this changes to a yellow 
brown. In the course of a week the growth is heavier and of a 
deeper brown color. Bouillon cultures show after a day in the 
incubator a somewhat intense cloudiness, with a moderate precipitate. 
No pellicle is formed on the surface. No indol reaction is present. 
Litmus milk after twenty-four hours appears reddish; otherwise, 
however, it undergoes no change. The milk never coagulates. 

The bacillus is pathogenic for mice, rabbits, and guinea-pigs. 
It is agglutinated by the patient's blood serum, and it is interesting 
to note that this reaction is obtained only with cases definitely known 
to have been infected with the microorganism in question. 



278 THE FECES 

Isolation of Shiga's Bacillus from the Feces. — The fecal matter is 
collected on a sterile pad, or, still better, obtained from the rectum 
by curettage. A bouillon culture is prepared and from this agar 
tubes are inoculated as soon as possible. The agar should be just 
acid to phenolphthalein (slightly alkaline to litmus), and is plated 
at once. Ten plates, variously diluted, are conveniently used. After 
twenty-four hours in the incubator at 37° to 38° C. all colonies are 
marked on the plates which have developed by that time. The 
plates are returned to the incubator. After further twenty-four 
hours tubes of glucose agar and litmus-mannite agar are inoculated 
from those colonies which have grown in the second twenty-four 
hours — i. e., those colonies which have not been marked. At the 
end of another twenty-four hours in the incubator all those tubes 
are rejected in which fermentation has taken place. From those 
tubes in which this has not occurred, litmus milk, litmus mannite, 
and bouillon are inoculated. The Shiga bacillus will at first render 
the milk slightly acid, but later it becomes alkaline. Litmus mannite 
remains unchanged with the Shiga strain, while the Flexner-Harris 
type (the American acid type) turns it red. Ultimate identification 
is made by the agglutination test in various dilutions (1 to 50 to 1 to 
100) reading the results after two hours. 1 

Literature. — K. Shiga, Centralbl. f. Bakt., Parasit. u. Infectionskrankh., 
1898, vol. xxiv. R. P. Strong and Musgrave, " Preliminary Note regarding the 
^Etiology of the Dysenteries of Manila," Report of the Surgeon-general of the 
Army, Washington, 1900, p. 251. S. Flexner, "On the Etiology of Tropical 
Dysentery," Bull. Johns Hopkins Hosp., 1900, p. 231. Vedder and Duval, "The 
Etiology of Acute Dysentery in the United States," Jour. Exper. Med., vol. vi, 
p. 181. Duval and Bassett, Amer. Med., 1902 ; iv, p. 417 (preliminary report). 

Bacillus typhi, Eberth. — The typhoid bacillus can only be demon- 
strated in the feces by cultural method which will enable its 
separation from other members of the colon group. To this end 
many different methods have been suggested. 

Combined Malachite-green Method of Lentz and the Method of 
v. Drigalski and Conradi. — This method is probably the most use- 
ful and extensively employed abroad. The media are prepared 
as described elsewhere (see Media). Two plates of Drigalski's 
medium are prepared in large Petri dishes, using 20 to 25 c.c. 
of the medium for each plate (15 to 20 cm. diameter); these are 
left uncovered until the steam has evaporated and the agar is quite 
firm. Contamination by the organisms of the air does not occur 
owing to the presence of the cresyl violet in the medium. The 
malachite-green medium is already plated when made (see Media). 
The stool is stirred up well with a small amount of sterile normal 

1 For a detailed account of the different varieties of the dysentery bacillus 
and dysentery-like organisms see J. C. Torrey, Journ. Exper. Med., 1905, vol 
vii, p. 365. 



BACTERIOLOGY OF THE FECES 279 

salt solution. Of this material about 0.5 c.c. is placed on the green 
plate and smeared over its surface with a glass rod, which is con- 
veniently bent at an angle about one inch from the end. Without 
sterilizing, the same rod is then smeared over the first Drigalski plate 
and hence over the second. After this all three are allowed to become 
perfectly dry by standing open in the air, when they are incubated 
for twenty to twenty-four hours. Plates 2 and 3 are now exam- 
ined with a hand lens, placing them, if possible, in such a position 
that light reflected from a wall falls upon them. The colon colonies 
are more or less red in color, not transparent, and measure 1 to 3 mm. 
in diameter. The typhoid colonies are bluish with a violet shade and 
resemble drops of dew. If such are found they are further identi- 
fied as follows: A tiny bit of the colony is placed on a slide and mixed 
with a drop of a highly active hundredfold dilution of typhoid (viz., 
paratyphoid) serum. Agglutination may be observed with a hand lens 
or a low power of the microscope. If this occurs further tests are 
made by inoculating ordinary agar, litmus whey, and neutral red agar 
(see Culture Media). 

If no colonies are found on the Drigalski medium which resemble 
typhoid bacilli, the green plate is flooded with sterile normal salt 
solution, gently agitated, and set aside for a few minutes. In this 
manner the typhoid and paratyphoid colonies, which are more delicate 
than the colon colonies, come to be disseminated in the fluid, while 
the latter sink to the bottom. With the glass spatula two more 
Drigalski plates are then prepared from the salt solution, incubated 
for twenty to twenty-four hours, and examined as described. 

If urine is to be examined in the place of feces, several drops are 
placed on the green plate and one drop only on the Drigalski plate. 
The procedure otherwise is the same. 

Blood is examined in a similar way, but must first be diluted in 
sterile bouillon to eliminate the bactericidal substances that are pres- 
ent (5 to 200). 

In pure cultures the typhoid bacilli present the following features : 
They occur in the form of rods of almost one-third the size of a red 
blood corpuscle, or in threads composed of several rods joined end to 
end (Figs. 76 and 77). Their ends are rounded; their length is equiva- 
lent to about three times their breadth. They are actively motile and 
provided with polar as well as lateral flagella. They grow very 
readily on bouillon-peptone gelatin, and after twenty-four hours 
colonies begin to appear. When slightly magnified, these present a 
faintly yellowish color; macroscopically they are barely visible. The 
organism does not form spores, but when kept at a temperature of 
37° C, and especially when grown on media colored with phloxin red 
or benzopurpurin, polar bodies are observed which were formerly 
mistaken for spores. Gelatin is not liquefied; the growth is white 
and delicate, both along the line of the stab and on the surface. Culti- 



280 THE FECES 

vation in glucose bouillon, or glucose agar, does not give rise to the 
formation of gas, but after twenty-four hours the entire fluid becomes 
turbid. Milk is rendered feebly acid, but is not coagulated. No 
indol reaction is obtained when the organism is grown on peptone- 
containing media. On potato a very faint, whitish, almost invisible 
growth takes place. When grown on gelatin or agar that has been 
colored with neutral red, the typhoid bacillus causes no change in 
color. Absolute identification is possible by means of Pfeiffer's 
agglutination test (see Widal's reaction). 

In cases of paratyphoid infection the corresponding organism may 
be found in the feces (see Blood). 

Bacillus acidophilus, Moro. 1 — This organism has been described 
by Moro as occurring in the stools of breast-fed infants, in which it 
normally predominates over all other forms; under pathological 





Fig. 76. — Typhoid bacilli from nutrient agar. Fig. 77. — Typhoid bacilli from nutrient 

X 1100 diameters. (Park.) gelatin. X 1100 diameters. (Park.) 

conditions, on the other hand, as also in the stools of children which 
have been fed with cows' milk, their number is found diminished, 
while the members of the coli group enter into the foreground. 
Beyond the stools the bacillus has been found in the outer portion 
of the secretory duct of the human mammary gland, in the milk, 
and the skin of the nipple and its immediate surroundings. It is 
apparently not pathogenic. 

The organism occurs in the form of slight rods measuring 1.5 p 
to 2 fi in length, by 0.6 fi to 0.9 fJ. in breadth. It is non-motile. 
It is not decolorized by Gram's method, but loses this property after 
from thirty-six hours to nine days. The best growths are obtained 
on beer-wort bouillon and common bouillon when acidified with a 
mineral acid; the acidity of 10 c.c. of the medium may correspond 
to 10 c.c. of a decinormal solution of potassium hydrate. The 
optimum temperature is 37° C; between 20° C. and 22° C. no growth 

1 " Ein Beitrag zur Kenntniss der normalen Darmbacterien des Sanglings," 
Jahrbuch f . Kinderheilk., vol. lii. Also : " Ueber die nach Gram farbbaren Bacillen 
d. Sauglingstuhles." Wien. klin. Woch., 1900, No. 5. 



BACTERIOLOGY OF THE FECES 281 

occurs. On the various agar slants imperfect development takes 
place; on potato the organism does not grow. It is an active acid 
producer, but does not give rise to the formation of gas ; with Escherich's 
stain it is colored blue. 

Escherich's Stain. — This stain is now extensively used by pediat- 
rists in order to ascertain any deviations from the normal in the flora 
of the feces. Under strictly normal conditions the bacilli which are 
found in the stools of breast-fed children are thus nearly all colored 
blue (these are essentially the anaerobic Bacillus bifidus communis, 
and the aerobic Bacillus acidophilus), while red bacilli (Bacillus coli 
communis and Bacillus lactis aerogenes) are but little numerous. In 
the case of infants, on the other hand, which are fed exculsively on 
cows' milk, the red bacilli predominate, while in mixed feeding the 
blue enter into the foreground in about the proportion in which breast 
milk is employed. The red bacilli belong to the coli group. These 
further predominate, or may be found exclusively, if for any reason 
intestinal digestion is impaired. Staphylococci, streptococci, etc., 
when simultaneously present, are in either event stained blue. In 
staphylococcus enteritis the blue bacilli which normally exist in the 
stools of breast-fed infants are almost entirely replaced by staphylo- 
cocci. At the beginning of the enteritis they are not numerous, but 
they increase during the progress of the disease, and finally disappear 
when the child recovers. 

In staining, the following solutions are employed: 

1. An aqueous solution of gentian violet (5 to 200). This is boiled 
for one-half hour and is then filtered; it keeps for a long time. 

2. A mixture containing 11 parts of absolute alcohol and 3 parts 
of oil of anilin. 

1 and 2 are mixed in the proportion of 8.5 to 1.5; the resulting 
solution keeps for from two to three weeks, but not longer. 

3. A solution of iodopotassic iodide containing 1 part of iodine 
and 2 parts of potassium iodide in 60 parts of water. 

4. A mixture of equal parts of oil of aniline and xylol. 

5. A concentrated alcoholic solution of fuchsin, diluted with an 
equal volume of absolute alcohol. 

A bit of the stool is spread upon a slide in as thin a layer as possible. 
After drying in the air the specimen is fixed by passing through the 
flame of a Bunsen burner. It is then stained for a few seconds with 
the mixture of 1 and 2, blotted, placed in the iodine solution for 
a few seconds, blotted again, decolorized with 4 until a notable 
extraction of color no longer occurs. It is washed with xylol, dried, 
and finally stained for a few seconds with the fuchsin solution, washed 
with water, blotted, and is then ready for examination. 

Bacillus (Proteus) vulgaris, Hauser. — This organism, while usually 
regarded as non-pathogenic, should be numbered among the bacteria 
which may at times develop pathogenic properties. Baginsky and 
Booker have frequently found it in the stools in cases of infantile 



282 THE FECES 

summer diarrhea. Escherich observed it at times in the meconium. 
Brudzinski examined the dyspeptic and fetid stools of a number 
of artificially fed infants in Escherich's clinic, and in all the cases 
found the proteus. Others have encountered it in inflammatory 
conditions of exposed surfaces, in appendicitis, in perforative peri- 
tonitis, and even in closed abscesses, either alone or in association 
with other bacteria (Welch). A mixed infection of the proteus with 
Lofner's bacillus has also been observed. The organism forms rods, 
measuring about 0.25 fi in diameter, while their length is variable; 
at times a more roundish form is observed; at others rods measuring 
from 1.25 // to 3.75 // in length, or even long threads. They are 
readily stained, but are easily decolorized by alcohol or Gram's method. 
Most characteristic is their growth upon nutrient gelatin. At the 
temperature of the room little depressions will be observed after six 
to eight hours, which are surrounded by a narrow zone of bacilli 
from which a thin, wide film, provided with irregular projections, 
extends over the culture medium. From this film islets become 
separated, which slowly extend over the gelatin and cause its lique- 
faction. The organism is motile. It decomposes urea and causes 
albuminous putrefaction. The nitroso-indol reaction is readily ob- 
tained in bouillon cultures. In boiled milk the organism grows well, 
while in fresh milk it develops only irregularly, and in acid milk no 
growth takes place at all. 

Bacillus pyocyaneus. — The Bacillus pyocyaneus has repeatedly been 
isolated from the stools of dysenteric patients, and has been proved the 
cause of several epidemics. The organism in question is a small 
motile bacillus measuring from 1 t a to 2 t a in length by 0.3 ju to 0.5 y. 
in breadth. It sometimes occurs in short chains, but is usually single. 
It is stained with the common aniline dyes, and is decolorized with 
Gram's method. It grows on the usual culture media, and liquefies 
gelatin. In 2 per cent, glucose bouillon no fermentation takes place. 
Litmus milk is curdled in about forty-eight hours. Some varieties 
produce indol. Most characteristic is the production of certain 
pigments, viz., pyocyanin and a fluorescent, bluish-green pigment 
which is common to almost all varieties. 1 

The Bacillus coli communis, 2 while constantly present in normal 
feces, is described at this place, as modern investigations have shown 
that it may at times develop pathogenic properties. It has been 
found in the pus in cases of purulent perforating peritonitis, angio- 
cholitis, pyelonephritis, etc.; it is frequently found infecting the 
bladder and the pelvis of the kidney, and, as indicated elsewhere, at 
times forms the nucleus of gallstones. It occurs in the form of deli- 
cate or coarse rods, measuring about 0.4 fi in length, which manifest 
a certain degree of motility, due to the presence of one or two polar 

1 A. J. Lartigau, " A Contribution to the Study of the Pathogenesis of the Bacillus 
Pyocyaneus," etc., Jour. Exper. Med., 1898, No. 6. 

2 Fliigge, Die Microorganismen. 






BACTERIOLOGY OF THE FECES 283 

flagella. The organism is stained by the usual aniline dyes, and is 
decolorized by Gram's method. The colonies upon gelatin closely 
resemble those of the bacillus of typhoid fever, forming small whitish 
specks in the gelatin, and delicate films with serrated borders upon 
the same medium, which, moreover, is not liquefied. On potato the 
organism forms a brownish pellicle, w^hile the growth of the typhoid 
bacillus is nearly transparent. As in the case of the cholera bacillus, 
the nitroso-indol reaction can be obtained when the organism is 
grown upon peptone-containing media. 1 In solutions of glucose 
active fermentation takes place. Litmus milk is rendered acid and 
is coagulated. Important also is the behavior of the organism 
when grown on gelatin or agar that has been colored with neutral 
red; in contradistinction to the typhoid bacillus, the colon bacillus 
then causes an intense green fluorescence. 

The Bacillus lactis aerogenes (Escherich) closely resembles the 
organism just described, and may also at times develop pathogenic 
properties. It is seen quite constantly in the stools of sucklings, but 
may also be met with in those of adults. It occurs in the form of 
rather stout rods, which. frequently He in pairs, resembling diplococci. 
The organism is non-motile. Like the Bacillus coli communis, it is 
decolorized by Gram's method. In plate cultures it forms a dense 
white film; in stab cultures a chain of white colonies resembling beads 
is seen. In the latter, moreover, if the stab is closed, bubbles of gas 
will be seen to form, which rapidly increase in number and size. 
Milk is coagulated in large lumps in twenty-four hours ; at the same 
time the formation of gas is much more intense than in the case of 
the Bacillus coli communis. 

The Comma Bacillus. — The first detailed studies of the organisms 
found in cholera stools were made in 1883 by the members of the 
French and German commissions sent to Egypt to investigate the 
nature of the dreaded disease. The results of their work were first 
published by Koch in his report to the Berlin Sanitary Office in 1883, 
and in 1884 by Strauss, Roux, Nocard, and Thaillier. 

The clinical recognition of cholera Asiatica has now become a 
simple matter since PfeifTer has demonstrated that the blood serum 
of cholera patients possesses the property of causing arrest of motility 
and agglutination of the specific bacilli. Ordinary bouillon cul- 
tures, however, can usually not be employed, as particles of the 
film when broken up may easily be mistaken for agglutinated bacilli. 
It is best in every case to make use of agar cultures sixteen to twenty- 
four hours old, and to prepare emulsions in bouillon. or normal salt 
solution as occasion requires. The emulsion, moreover, should always 
be examined microscopically before use, so as to ensure the absence of 

1 The test for indol is very conveniently made by adding a few drops of Ehr- 
lich's dimethyl-amino-benzaldehyde solution (see Urine) to a culture of the 
organism in Dunham's solution which has grown for four or five days. On 
shaking, and especially on heating, a cherry-red color develops. 



284 THE FECES 

any conglomeration of bacilli. The blood is then diluted in the pro- 
portion of 1 to 10 or 1 to 15. If the test-tube method is employed, the 
tubes should be kept in the incubator (37° C.) for only one or two 
hours. Upon the slide the reaction is obtained in from five to twenty 
minutes. If no distinct agglutination is observed at the end of one 
hour, the diagnosis of cholera is rendered improbable. Dried blood 
retains its agglutinating properties for a considerable length of time, 
and may also be used for examination. 

The comma bacillus is a slightly arched or half -moon-shaped 
little rod, and is somewhat shorter than the tubercle bacillus (Fig. 
78). Occasionally two are placed end to end with their convexities 
in opposite directions, thus presenting the appearance of the letter 
S. They are provided with flagella. Koch detected these bacilli 
in the intestinal contents and feces, but rarely in the vomited matter, 
in Asiatic cholera only. In the stools they at times occur in such 




Fig. 78. — Cholera spirilla preparation from gelatin-plate culture of cholera. 
X 800 diameters. (Park.) 

numbers as to constitute pure cultures. In plate cultures kept at 
a temperature of 22° C. white colonies with serrated borders may be 
observed after twenty-four hours. The color of such a colony is 
slightly yellow or rose red, its central portion gradually assuming a 
deeper tint, and finally becoming liquefied. Upon agar plates the 
bacilli form a grayish-yellow, irregular, slimy coating, but do not 
liquefy the culture medium. In stab cultures, after twenty-four hours, 
a whitish color may be observed along the line of the stab; around 
this there is found a funnel-shaped depression, which gradually 
increases in size and apparently contains a bubble of gas. The upper 
portion of the culture medium at the same time becomes liquefied 
while the lower portion remains solid for days. In a suspended drop 
spirochete-like spirals are observed at the margins, which often pre- 
sent as many as twenty distinct arches 1 

1 R. Koch, Berlin, klin. Woch., 1884, vol. xxi, pp. 477, 493, 509. 



ANIMAL PARASITOLOGY OF THE FECES 285 

Closely related to Koch's comma bacillus is the bacillus of Finkler 
and Prior, 1 discovered in 1884 and 1885. It is distinguished from 
the former by the following characteristics: it is larger and thicker 
than the comma bacillus ; the colonies on gelatin plate cultures show 
equally round and sharp-edged forms, which present a granular 
appearance under a low or medium power, and are usually of a brown 
color. The organism liquefies gelatin very rapidly, a penetrating, 
excessively fetid odor being developed at the same time. In stab 
cultures the bacillus of cholera Asiatica forms a funnel-shaped depres- 
sion, while the bacillus of Finkler and Prior forms a stocking-like 
depression. 

Tubercle bacilli, when present in the feces, are indicative of intes- 
tinal tuberculosis, providing they are observed upon repeated exami- 
nation and there are clinical symptoms pointing to the bowels as the 
seat of the disease; otherwise they may be referable to swallowed 
sputa. They may be demonstrated as described in the chapter on 
Sputum. 



ANIMAL PARASITOLOGY OF THE FECES. 

The animal parasites which may be met with in the feces are classi- 
fied as follows: 

I. — Protozoa : 

1. Rhizopoda, 

Monera, 

Amoebina : Amoeba coli. 

2. Sporozoa, S. gregarina, 

Coccidia, 

3. Infusoria, 

a. Ciliata, 

Holotricha: Balantidium coli. 

b. Flagellata. 

Monadina, 

Cercomonadina : Cercomonas. 
Isomastigoda. 

Tetramitina : Trichomonas. 
Polymastigina : Megastoma. 
II. — Vermes: 

Platodes, 

Cestodes, 

Taenia saginata. 
Taenia solium. 
Taenia nana. 
Taenia diminuta. 
Taenia cucumerina. 
Bothriocephalus latus. 
Krabbea grandis. 

1 Finkler, Deutsch. med. Woch., Tageblatt der Naturf orscherversammlung, 1884, 
vol. x, p. 36, and 1885, p. 438. Finkler u. Prior, Erganzungsheft z. Centralbl. f. 
allg. Gesundheitspflege, 1885, vol. i. 



286 THE FECES 

Trematodes, 

Distoma hepaticum. 
Distoma lanceolatum. 
Distoma Buskii. 
Distoma sibiricum. 
Distoma spatulatum. 
Distoma conjunctum. 
Distoma heterophyes. 
Amphistoma hominis. 
Distoma haematobium. 
Distoma pulmonale. 
Annelides, 

Nematodes, 

" Ascarides, 

Ascaris lumbricoides. 

Ascaris mystax. 

Ascaris maritima. 

Oxyuris vermicularis. 
Strongyloides, 

Ankylostomum duodenale. 
Trichotrachelides, 

Trichocephalus hominis. 

Trichina spiralis. 
Rhabdonema strongyloides, 

Anguillula intestinalis. 

Protozoa. — The rhizopoda are essentially characterized by the fact 
that locomotion does not take place by the aid of independent organs, 
but by means of pseudopodia, viz., protoplasmic processes which the 
animal is capable of protruding from any portion of its body. Six 
orders have been described by zoologists, but only one, or possibly 
two, have thus far been found in the feces. 

Whether or not representatives of the monera occur in the feces of 
man is still an open question. If so, they are apparently of no 
pathological significance. 1 

Of the amoebina, on the other hand, a most important member has 
been found, viz., the Entamoeba dysenteric. 

Entamoeba Dysenterise, S. Histolytica (Schaudinn): syn., Amoeba 
Coli (Losch). — In 1875 Losch 2 discovered in the stools of dysenteric 
patients actively moving cell-like bodies of a roundish, pear-shaped, 
oval, or irregular form. He did not regard these as the cause of the 
disease, however, but looked upon them as only accidentally present. 
Similar bodies were observed in Hong-Kong by Normand in cases 
of colitis; and also by v. Jaksch. Sansino found them in a case in 
Cairo, and Koch in East Indian dysentery. It is interesting to note 
that Koch was the first to suspect the existence of a definite relation 
between dysentery and these organisms. Cunningham claims to 
have found amebas frequently in the stools of cholera patients at Cal- 
cutta, and Grassi in normal stools, but especially abundant in cases 

1 Nothnagel, loc. cit., p. 110. Grassi, cited by Bizzozero. v. Jaksch, Wien. 
klin. Woch., 1888, vol. i, p. 511. 

2 "Massenhafte Entwickelung v. Amoben im Dickdarm," Virchow's Archiv, 
vol. lvi. 



ANIMAL PARASITOLOGY OF THE FECES 287 

of chronic diarrhea. Whether all these observations are correct, and 
whether the organisms observed were identical in all cases, is, of course, 
difficult to say. So much is certain, that the subject was still in a very 
unsettled state when Kartulis 1 announced " that dysentery and tropi- 
cal liver abscess associated with dysentery are caused by the presence 
of the Amoeba coli," basing his conclusion upon an examination of 
500 cases. The fact that this parasite was absent in all other 
intestinal diseases, such as typhoid fever, intestinal tuberculosis, the 
ordinary forms of diarrhea, etc., speaks stmngly in favor of Kartulis' 
view. 9 

In perfect accord with these observations are those made at the 
Johns Hopkins Hospital. 2 Osier 3 was the first in this country to 
demonstrate the presence of the Amoeba coli in a case of liver abscess, 
both in the pus of the abscess and in the stools. Stengel, Musser, 
Dock, and others confirmed these observations, and the pathogenic 
character of the Amoeba coli may now be regarded as an established 
fact. 4 This statement is based not only upon the few facts, more his- 
torical in character than otherwise, which have just been detailed, 
but rather upon the ensemble of collected data, among which the 
absence of microorganisms other than the ameba in the pus of the 
liver abscesses, and the constant presence of the latter in such cases, 
rank among the most important. It is to be noted, however, that 
different forms of tropical dysentery exist, and that the Amoeba coli 
is essentially associated with the more chronic form, while the acute 
types are of bacillary origin (see Shiga's bacillus). 

The size of the amebas averages 35 fi. When at rest their outline 
is, as a rule, circular, occasionally ovoid; but when in motion they 
present the extremely irregular contour of moving ameboid bodies 
(Plate XIV). The protoplasm can be differentiated into a trans- 
lucent, homogeneous ectosarc or mobile portion, and a granular 
endosarc, containing the nucleus, vacuoles, and granules. Within 
the endosarc the vacuoles constitute the most striking feature. Some- 
times the interior seems to be made up of a series of closely set, clear 
vesicles of pretty uniform size. As a rule, one or two larger vacuoles 
are present, the edges of which are not infrequently surrounded by 
fine, dark granules. True contractile vesicles displaying rhythmic 
pulsations have not been observed, although the vacuoles may at 
times be seen to undergo changes in size. In some the nucleus is 
quite distinct, while in others it may be altogether invisible. The 
protoplasm of the amebas is strongly basophilic. 

1 "Zur Aetiologie d. Dysenterie in Egypten," etc., Virchow's Archiv, 1885, vol. 
cv, and 1889, A 7 ol. cxviii. Centralbl. f. Bakt. u. Parasit., 1890, vol. vii. 

2 Councilman and Lafleur, "Amoebic Dysentery," Johns Hopkins Hosp. Rep., 
1891, vol. ii. C. E. Simon, Johns Hopkins Hosp". Bull., 1890. 

3 Johns Hopkins Hosp. Bull., 1890. 

4 For the more recent literature see especially H. F. Harris, "Amoebic Dysen- 
tery," Amer. Jour. Med. Sci., 1898, p. 384. 



288 THE FECES 

Most distinctive are the movements of these bodies. From any 
part of the surface a rounded, hemispherical knob will project, and 
with a rapid movement the process extends and the granules in the 
interior flow toward it. In these movements the clear ectosarc seems 
to play the most important part. The organisms are actively phago- 
cytic and often contain red corpuscles, bacteria, and crystals. Repro- 
duction occurs by fission. 

Various attempts have been made to cultivate the Amoeba coli, but 
on the whole the results have not been satisfactory. In every attempt 
in this direction adequatS bacterial symbiosis must be secured. The 
most comprehensive work in this direction has been done by Musgrave 
and Clegg. The medium which they recommend has the following 
composition and is prepared as ordinary agar: 

Agar 20.0 j 

Sodium chloride . 3-0 . 5 \ pro liter. 

Beef extract 0.3-0.5 J 

The final product is most universally satisfactory when 1 per cent, 
alkaline to phenolphthalein, to which end it is recommended to start 
with an initial alkalinity of 1.5 per cent. 

Tubes of this medium are plated and the surface lightly smeared 
with material selected from feces containing amebas. The first plates 
must be watched frequently under the microscope, and as soon as it 
is found that amebas have developed (twenty-four hours to four or 
five days) transplants must be made, as otherwise they are liable to 
die. For further details to this end the reader is referred to Mus- 
grave and Clegg's monograph. 1 

To demonstrate amebas in stools it has been generally suggested to 
procure bits of mucus or mucopus for examination. Musgrave and 
Clegg recommend that the patient be given a saline cathartic and that 
the examination be made from the fluid portion of the stool. Drops 
of this are mounted, covered with cover-glasses, and examined with 
a \. The diagnosis of amebiasis should then only be made if motile 
amebas are encountered. Resting or encysted forms may be mistaken 
for epithelial cells, swollen leukocytes, etc. 

Not infrequently some of the organisms are found containing one 
or more red cells. (Plate XIV.) 

Staining is not at all essential for the purpose of demonstrating 
amebas in the stool. The examination of the fresh material is much 
more satisfactory and far less likely to lead to errors of diagnosis. 

Very pretty pictures are obtained by vital staining with neutral red. 
(Plate XIV.) To this end it is only necessary to run a drop of a 
dilute solution of the dye under the cover-glass, when it will be seen 
that the young, actively motile amebas take up the stain without 

1 Amebas, Bureau of Government Laboratories. Biological Laboratory of 
Manila, 1904. 



PLATE XIV 






t 2 



4 

« 



;K..;4s ' '*■ - : - 



ft# 



Amoebae Fed with Neutral Red and Containing 
Phagocytes and Red Cells. 



PLATE XV = 









Eggs of Parasites. 

a, Uncinaria americana ; b, Trichocephalus dispar ; c, Oxyuris vermicularis ; d, Tsenia saginatt 



ANIMAL PARASITOLOGY OF THE FECES 289 

losing their motility. They can then be readily watched in their 
movements. 

The preparation of stained permanent preparations is not very 
satisfactory. They are prepared like blood films and colored with one 
of the modifications of the Romano wsky dye. 

When older material only is available it may be difficult to arrive 
at a satisfactory conclusion. Sometimes it is possible to cause the 
amebas to move again by warming the stool in an open dish at body 
temperature, but more often they are dead. Attention should then 
be especially directed to ameba-like structures containing red blood 
cells. If such are found the inference that the cell is a dead ameba 
is usually warrantable. 

Entamoeba coli (Schaudinn). — This is not to be confused with the 
Entamoeba dysenterise. It is smaller than the Entamoeba dysenteria?, 
the size varying between 10 and 15 (J-. It is opaque, gray in color, 
and provided with a distinct nucleus. The ectoplasm is usually not 
visible. The movements are much more sluggish and the tendency 
to phagocytosis much less marked. It is considered to be non-patho- 
genic. In the Philippines it is apparently quite common. Craig 1 
finds 65 per cent, of normal individuals infected with it, but uses 
saline purgatives to produce diarrheal discharges, as recommended 
by Musgrave. 

Paramoeba hominis (Craig). — Craig 2 observed organisms which 
apparently occupy a position intermediary between amebas and flagel- 
lates, in several cases of severe diarrhea occurring in the Philippine 
Islands. In one stage of its existence the parameba is capable of active 
progressive locomotion and is much larger than the trichomonas in 
the resting stage. In the flagellate stage it is distinguished from the 
corresponding stage of trichomonas by the absence of an undulating 
membrane, the presence of a single flagellum, and its circular form. 
The question of its pathogenicity has not been decided. 

The Flagellata s. mastigophora differ from the rhizopoda in being 
provided with from one to eight flagella, which serve as organs of 
locomotion and possibly also for the apprehension of food particles. 
Representatives of two orders only, viz., the monadina and isomasti- 
goda, have been found in the feces. Of the monadina in turn only 
one family, viz., the cenomonadina, and of the isomastigoda only 
two families, the tetramitina and polymastigina, are represented. 3 

The cenomonadina are small, oval, frequently elongated bodies, 
provided with one long flagellum at the anterior end, at the base of 
which food vacuoles are situated. At the posterior end ameboid 
movements may be observed, and there can be no doubt that the 

1 Amer. Med., 1905, pp. 850, 897, and 936. 

2 Amer. Jour. Med. Sci., August, 1906, p. 214. 

3 W. Janowski, Zeit. f. klin. Med., vol. xxxi, p. 445. 
19 



290 THE FECES 

taking up of food, to some extent at least, also occurs by the aid of 
pseudopodia. To this family belongs the cercomonas of Davaine 
and Lambl. The tetramitina are small, elongated bodies, provided 
with four flagella and a lateral, undulating membrane, which was 
formerly mistaken for a posteriorly directed flagellum. The tail 
end of the organism tapers to a point. The nucleus is located at 
the base of the flagella. To this family belongs the parasite which 
was first discovered by Donne in the vagina, and which later was 
found also in the feces, and which has been variously designated as 
Trichomonas hominis, Cercomonas coli hominis, etc. 

The polymastigina are small, somewhat oval bodies, provided with 
two or three flagella, situated either anteriorly or laterally — two or 
three on each side — while at the same time two additional flagella 
issue from the posterior end, which may either be rounded off or 
taper to a point. 'To this family belongs the Megastoma entericum 
of Grassi. 

The question whether or not the flagellate bodies are of patho- 
logical importance still remains sub judice. They are apparently 
met with only in diseases associated with diarrhea, and it appears 
that in some cases at least this is directly dependent upon their pres- 
ence; in others the impression is gained as though they merely main- 
tained an already existing diarrhea referable to other causes ; while in 
a third class of cases no relation can be discovered between their pres- 
cence and the disease in question. Cohnheim 1 has pointed out that 
living infusoria in the feces may be a symptom of a primary chronic 
stomach affection (gastritis, usually the atrophic form). According 
to the same writer, encysted infusoria may also be found in the feces 
of healthy individuals, but in such cases we may assume that at some 
time previously a gastritis or a gastro-enteritis has existed. He thinks 
they have no pathogenic significance, and are merely of symptomatic- 
diagnostic interest. 

Cereomonas of Davaine-Lambl : syn., Cercomonas hominis (Da- 
vaine); monas (Marchand); Monas lens (Grassi); Monas mono- 
mitina (Grassi). The adult organism (see Fig. 79) is oval or roundish 
in form, and provided anteriorly with a single long flagellum and 
posteriorly with a tail-like appendage. Its length varies from 0.005 
to 0.014 mm. The younger forms are pear-shaped or S-shaped, 
and sometimes irregular in outline ; the flagellum is then either absent 
or rudimentary. 

Upon prolonged observation it will be seen that the adult parasite 
loses its flagellum and may protrude a protoplasmic process instead, 
while vacuolation occurs at the same time, indicating approaching 
death. 2 

1 Deutsch. med. Woch., 1903, vol. xxix, p. 248. 

2 Lambl, Prag. Vierteljahr., 1859, vol. lxi, p. 1. Davaine, Traite des entozo- 
aires, 1860, Paris. Marchand, Virchow's Archiv, 1875, vol. lxiv, p. 293. Zunker 
Deutsch. Arch. f. prakt. Med., 1878. 



ANIMAL PARASITOLOGY OF THE FECES 



291 



Trichomonas, Donne: syn., Trichomonas vaginalis (Donne); 
Trichomonas hominis (Grassi); monocercomonas (Grassi); cimseno- 




Fig. 79.— Cercomonas intestinalis: a, Cercomonas of Davaine, after Leuckart; b, Cerco- 
monas intestinalis, after Lambl; c, d, same, ordinary forms: e, f, same, well-developed forms; 
g, h, i, same.Jdegeneration forms; k, I, same, abortive forms. 




Fig. 80.— Trichomonas intestinalis: a, a', c, trichomonas of the urine, after Marchand; b, 
Trichomonas vaginalis, after Donne; d, Trichomonas intestinalis, after Piccardi; e, e', e", 
same, ameboid forms; f, f, trichomonas of the urine. (After Dock.) 

monas (Grassi) ; Protorycomyces coprinarius (Cunningham and Lewis) ; 
Cercomonas coli hominis (May); Trichomonas intestinalis (Leuckart 



292 



THE FECES 



and Roos); Cercomonas s. Bodo urinarius (Kunstler). The parasite 
(Fig. 80) is oval or spindle-shaped and measures from 0.012 to 0.03 
mm. in length by 0.01 to 0.015 mm. in breadth. From its anterior 
pole four flagella are given off, which are almost as long as the 
organism itself. From this point an undulating membrane extends 
laterally to the posterior pole, which may be rounded off or tapers to 
a tail-like appendage. This membrane is best seen when the move- 
ments of the flagella have ceased, as in specimens fixed in mercuric 
chloride solution (1 to 5000). The nucleus is situated at the base 
of the flagella, but is usually visible only in stained specimens (methy- 
lene blue). At times the organisms may be observed to assume an 
ameboid form; the movements of the flagella have then ceased, and 
pseudopodia-like processes are protruded. The parasite is identical 






Fig. 81. 



-Megastoma entericum: a, front view; b, side view; c, organism attached 
to an epithelial cell. (Mosler.) 



with the trichomonas which has been found in the vagina and in 
the urine. 1 When present in the feces the organism is usually seen 
in large numbers. Not infrequently it is found associated with other 
intestinal parasites. 

Megastoma entericum, Grassi: syn., Cercomonas intestinalis 
(Lambl); Megastoma intestinale (Butschli); Lamblia intestinalis 
(Blanchard); Dimorphus muris (Grassi). The parasite (Fig. 81) 
is pear-shaped, and measures from 0.01 to 0.021 mm. in length by 
0.0075 to 0.05 mm. in breadth. In its anterior portion a more or 
less well-marked depression can be made out, which constitutes the 
peristome or mouth-opening of the organism. It is provided with 



1 Marchand, loc. cit. Zunker, loc. cit., p. 236. 
Spez. Path, u, Therap., 1894, vol. vi. 



Mosler u. Peiper, Nothnagel's 



ANIMAL PARASITOLOGY OF THE FECES 



293 



eight flagella, grouped in pairs. The first pair originates on the sides 
of the peristome and is directed backward. The second and third 
pair are situated somewhat posteriorly and are likewise directed 
backward, while the fourth pair issues from the tapering tail end of 
the body. In fresh specimens the eighth flagella can usually not be 
made out, as the third and fourth pair are frequently agglutinated. 
The best results are obtained when the organism has been killed with 
mercuric chloride solution. The individual flagella vary from 0.009 
to 0.014 mm. in length. In the anterior portion of the peristome 
two round, hyaline bodies can be recognized, which represent nuclei. 
Vacuoles are absent, and nutrition occurs through osmosis, the para- 
site adhering to epithelial cells by its periostome. When treated with 
fixing solutions the chitinous envelope can be readily recognized. In 
the encysted form the organism is oval and measures from 0.007 to 
0.1 mm. in diameter. 




Fig. 82. — Balantidium coli; 




division; 3, conjugation. (After Leuckart, from Doflein. 



Grassi observed the organism in mice, rats, cats, dogs, rabbits, and 
sheep. 1 The ciliata } as the term indicates, carry cilia, and of these 
only one member, belonging to the holotricha, is found in the feces, 
namely, the Balantidium coli. 

Balantidium coli, Stein: syn., Paramcecium coli (Malmsten). The 
organism is oval and measures from 70 ti to 110 fx in length by 60 
it to 72 fj. in breadth. It is covered entirely with fine, actively motile 
cilia, which are grouped most densely about the funnel-shaped mouth, 
while at the anus only a few are seen. An ectosarc and an endosarc 
may be distinguished, and the parasite possesses the power to change 
its shape, and may appear quite round. In its interior we find a 
large, somewhat kidney-shaped nucleus, two contractile vesicles, and 
frequently fat droplets, starch granules, etc. (Fig. 82). 



Grassi u. Schewiakoff, Zeit. f. wiss. Zoologie, 1888, vol. xlvi, p. 143. 



294 



THE FECES 



The parasite is probably pathogenic, but comparatively uncommon 
outside of Sweden, Finland, and Russia. Infection occurs through 
the dejecta of swine. Strong and Musgrave report that in their case 
blood examination showed a relative increase of the eosinophiles. 
From 200 to 300 organisms have been encountered in a single drop of 
the liquid feces. 1 

The fourth class of protozoa, viz., the Gregarina or sporozoa, 2 is 
also said to be represented in the human feces. The coccidia and 
psorosperms belong to this order. They are oval bodies, measuring 
about 0.022 mm. in length, and contain in their interior a large 
number of small nuclei arranged in groups. They are entirely 
devoid of organs of locomotion, and obtain their nutriment by 




Fig. 83. — Segments of tapeworms: a, Taenia saginata; b, Bothriocephalus latus; 
c, Taenia solium. 

endosmosis. Reproduction occurs in a common capsule, which 
bursts at a certain time and sends forth a whole generation of fully 
developed organisms. In human pathology they have become of 
interest in so far as certain observers have ascribed to them a role in 
the etiology of neoplasms. A disease of the liver analogous to the 

1 Malmsten, Virchow's Archiv, 1897, vol. xii, p. 302. Sievers, "Ueber Balanti- 
dium Coli im menschlichen Darmkanal," Arch. f. Verdauungskrank., vol. v, p. 
445. Janowski, " Balantidium Coli," Zeit. f. klin. Med., vol. xxxii, p. 303. 
Henschen, Arch. f. Verdauungsk., 1901, vol. vii, p. 501. Solorojew, Centralbl. f. 
Bacter., 1901, vol. xxix, pp. 821 and 849. A. Ehrenrooth, Zeit. f. klin. Med., 
1903, vol. xlix, p. 321. 

2 v. Wasielewski, Sporozoenkunde, 1896. 



ANIMAL PARASITOLOGY OF THE FECES 



295 



psorospermiasis of rabbits has also been described in man, and para- 
sites belonging to the same order have been observed in the skin. 

Cestodes. — T aenia saginata, Goeze : syn. , T. mediocanellata (Kiichen- 
meister) ; T. incruris (Huber) ; T. dentata (Nicola). This parasite (Fig. 
84) is the most common tapeworm in Europe and North America.- 






Fig 84.— Taenia saginata: a, natural size; b, head much enlarged; c, ova"much'enlarged. 

Infection occurs through the ingestion of measly beef. Its length 
varies from 4 to 8 m. The head, which is devoid of a rostellum, 
is surrounded by four pigmented suckers, each of which is encircled 
by a dark line. The individual segments are quite thick and opaque, 
and diminish in length as the head is approached, the largest measur- 
ing from 2 to 3 cm. They are each provided with a very much 



296 THE FECES 

branched uterus, which opens laterally, the primary branches num- 
bering about twenty on each side (Fig. 83). The ova are elliptical 
in form, of a brown color, and usually enclosed in a vitelline mem- 
brane (Plate XV) . Upon careful observation a double contour with 
delicate, radiating striae can be discerned. In the interior the hook- 
lets of the embryos, which are lost in the adult worm, are seen em- 
bedded in a brown, granular material. 

The diagnosis is mostly made by the patient when segments are 
found in the stools. In doubtful cases the eggs should be looked for; 
they are readily seen with a low power (J Bausch and Lomb). 

The larval form of Taenia saginata, the so-called Cysticercus taeniae 
saginatae (Leuckart), or the Cysticercus bovis (Cobbold), has been 
encountered in cattle, the Rocky Mountain "antelope," the llama, 
and the giraffe. In the human being it has not been observed. 1 

Taenia solium, Rudolphi: syn., T. cucurbitina, plana, pellucida, 
Goeze. This parasite (Fig. 85) is far less common in this country than 
the Tenia saginata, and may indeed be regarded as a curiosity. In 
Germany, also, it is only rarely met with now, while formerly it 




Fig. 85.— Head of T. solium. X 45. (Leuckart.) 

was the most common tapeworm in that country. This change is 
undoubtedly owing to the fact that raw pork is now eaten less fre- 
quently. In Asia and Africa it is more common. 

Taenia solium is usually much shorter than Tenia saginata, rarely 
exceeding 3.5 m. in length. Most characteristic is the head, which 
is provided with four pigmented suckers and a rostellum, furnished 
with from twenty-four to twenty-six hooklets arranged in a double 
row. The mature segments measure from 1 to 1.5 cm. in length 
by 6 to 7 mm. in breadth, and contain a uterus which has only five 
to seven branches, thus differing greatly from that of Taenia saginata. 
The ova are round, of a brownish color, and surrounded with a 
thick, radially striated membrane; in their interior the hooklets of 
the embryos can usually be made out. They are readily found in 
the feces and should be looked for in doubtful cases. 

1 J. Ch. Huber, Die Darmcestoden des Menschen. Bibliograph. d. klin. Helmin- 
thol., Heft 3, No. 4, p. 69, Miinchen, 1892. R. Leuckart, Die Parasiten des Men- 
schen, etc., 2d ed., 1880, pt. i. 



ANIMAL PARASITOLOGY OF THE FECES 297 

F The larval form of this tapeworm, the Cysticercus cellulosce, has 
been found in swine, the wild boar, in monkeys, in the brown bear, 
in the dog, etc. At times, though rarely, an auto-infection with the 
proglottides of Taenia solium has also been observed in the human 
being. Under such conditions the embryos of the worm are set free 
in the stomach, and may then migrate into various parts of the 
body, where they become encysted. Most commonly the cysticerci 
are found in the skin; they have, however, also been observed in 
the heart, the lymph glands, liver, bones, tongue, spinal canal, the 
brain and the eyes. I have had occasion to observe a case of this 
kind at the Johns Hopkins Hospital (reported by Osier). The 
patient, a laboring man, had never worked as a butcher or a cook, 
and never had a tapeworm. The cysticercus nodules, which were 
situated between the skin and the fascia, were very numerous, 
seventy-five being counted on one day. One of these nodules was 
removed for examination, and was shown to be referable to the 
cysticercus of Taenia solium. The only subjective complaints in this 
case were pains and stiffness in the arms and legs. The individual 
cysticercus was elliptical or roundish in form, measuring from 1 to 
10 mm. in diameter. In its interior the characteristic hooklets were 
seen. 1 

Taenia nana, v. Siebold: syn., hymenolepis (Weinland). This 
parasite (Fig. 86) seems to be the most common tapeworm of Italy 
and Egypt. It has also been seen in Buenos Ayres, in Bangkok, 
Siam, and a few isolated cases have been reported in England and 
in Germany. In the United States the parasite seems to be not at 
all uncommon, but has probably been overlooked in many cases. 
Stiles states that in his laboratory eighteen cases have been diagnos- 
ticated within a year (1902). It is found especially in young people, 
and often causes severe nervous symptoms. It is only 8 to 25 mm. 
long and 0.5 mm. broad. The head is ball-shaped and provided 
with four suckers and a rostellum, bearing twenty-four to twenty- 
eight hooklets arranged in a single row along its anterior edge. The 
individual segments are of a yellowish color and about four times as 
broad as long. The uterus is oblong and contains numerous ova, 
which are colorless, oval, and surrounded by a distinct, non-striated 
membrane. They measure from 0.839 to 0.060 mm. in size. In 
their interior the embryonic worm, provided with five or six hooklets, 
may be distinguished. The number of worms which may at times 
be found in the digestive tract is most astonishing; 5000 and even 
more have been counted on several occasions. The cysticercus stage 
occurs in snails, which are frequently eaten raw in Egypt and Italy. 
Taenia nana has been identified with the Taenia murina of rats and 

1 Huber, loc. cit. Leuckart, loc. cit.; and Blanchard, Traite de Zoologie 
medicale, vol. iv, Paris. The Inspection of Meats for Parasites, Bull. No. 19, 
Bureau of Animal Industry, Washington, 1898. 



298~ 



THE FACES' 











Fig. 86. — Taenia nana: 1, body; 2, natural size; 3, head; 4, booklets; 5, eggs; 6, egg 
magnified 600 times. (From Mosler.) 

other rodents. 1 In doubtful cases the eggs should be looked for; they 
are readily seen with a low power (B. and L. f). 

1 Grassi, Centralbl. f. Bakt. u. Parasit., 1887, vol. i, p. 97. Grassi u. Caland- 
ruccio, ibid., 1887, vol. ii, p. 282. Comini, ibid., p. 27. Bilharz, cited by Leuc- 
kart ' C W Stiles, New York Med. Jour., Nov. 7, 1903. 



animal Parasitology of The feces $$# 

Taenia dimimcta,, Rudolphi: sijn., Taenia flavapunctata (Weinfand); 
Taenia minima (Grassi)); Taenia varerina (Parona); Taenia lepto- 
cephala (Creplin). Taenia diminuta was first described in man by 
Leidy, Grassi, and Parona. It measures 20 to 60 mm', in length,, 
and is armed with two suckers* but is without a rosteftum. The 
ova resemble those of Taenia solium. The cysticercus^ occurs in- 
certain caterpillars and cocoons. In man it has^ beetf found in only 
six instances. 1 

Dipylidium caninum, Linne: syn., Taenia canina (Linne); Taenia; 
moniliformis (Pallas); Taenia cucumerina (Bloch); Taenia eiliptica 
(Batsch). The parasite is found almost exclusively in children; 
infection occurs through dogs and cats. In the United States the 
disease is apparently rare. The only case reported is that of Stiles. 2 
The larval form is found in lice and fleas. The worm itself measures 
from 15 to 35 cm. in length. The head is small, globular; the rostel- 
lum club-shaped with 3 or 4 transverse rows of hooks (about 60 
in number) of rose-thorn form ; anterior hooks 1 5 ju, posterior hooks 
6 fjt; suckers relatively large, rather elliptical. Segments 80 to 120 
in number; gravid segments 8 to 11 mm. long, 1.5 to 3 mm. broad; 
often reddish-brown in color. Genital pores at equator or in posterior 
half of segment; uterus forms egg capsules, eacji containing from 8 
to 20 eggs, eggs globular, 43 to 50 p in diameter. The ova contain 
embryos already armed with hooklets (Stiles). In diagnosis Stiles 
suggests that search be made in the feces for the peculiar elongated 
elliptical tapeworm segments (Fig. 87). Microscopic examination 
of the feces for eggs is less certain than in cases of infection with 
Taenia saginata, Taenia solium, or Dibothriocephalus latus, since 
Dipylidium is much smaller and less prolific than any of these three 
forms. 3 

Taenia Africana, v. Linstow. 4 — This parasite has been found in two 
instances, in the case of two native soldiers at Nyasa Lake. Like 
the scolex of Taenia saginata, that of the present species is devoid of 
hooklets. Its length is about 1.4 m. ; the number of segments about 
600. They are all much broader than long. The uterus consists of 
a main portion running fore and aft, from which from 15 to 24 side 
branches issue, which do not branch dichotomously and are so closely 
packed that they cannot be recognized with the naked eye. 

Taenia Madagascariensis (Grenet). — This parasite has been found in 
Madagascar, in Mauritius, in Bangkok, and in a Demarara Indian. 
The worm attains a length of from 25 to 30 cm. and is composed of 

1 Leidy and Parona, cited by Leuckart. 

2 Amer. Med., 1902, vol. v, p. 65. 

3 A. Hoffmann, Jahresb. f. Kinderheilk., 1887, vol. xxvi, Heft 3 u. 4. Kriiger, 
St. Petersburg, med. Woch., 1887, vol. xii, p. 341. Brandt. Centralbl. f. Bakt. u. 
Parasit., 1889, vol. v, p. 99. 

4 Centralbl. f. Bakt. u. Parasit., 1900, vol. xxviii, p. 485. 



300 



THE FECES 



from 500 to 600 trapezoid segments. The rostellum is surrounded by 
a double row of minute hooklets. The suckers are round and quite 
large. Blanchard suggests that the cockroach may be its interme- 
diary host. 

Dibothriocephalus latus, Linne, Lueke: syn., Bothriocephalus latus, 
(Bremser), Taenia lata (Linne); Dibothrium latum (Rudophi) (see 




Fig. 87. — a, Dipylidium caninum (taken from Stiles); b, gravid segment (after Diamare); 
c, head, showing four rows of rose-thorn hooks on the rostellum and four unarmed suckers 
(Stiles); d, egg, showing six hooks of the embyro (Stiles). 







Fig. 88. — Bothriocephalus latus: a, b, twin segments. (Wilson.) 



Fig. 88). This worm is usually 5 to 10 m. long and of a reddish- 
gray color. Longer specimens, however, may also be encountered. 
In Wilson's case 82 feet of segments were obtained from two worms, 
so that the length of each, supposing both to have been of the same 
size, must have been more than 40 feet. The head is almond-shaped 



ANIMAL PARASITOLOGY OF THE FECES 301 

and upon its flat surfaces two distinct grooves can be discerned, which 
probably act as suckers. It measures 2 to 3 mm. in length by 1 mm. 
in breadth. The neck is very short and passes at once into the body 
segments. Adjacent segments can often be distinguished only by 
means of the recurrence of the sexual apparatus, which appears 
regularly in spite of the imperfect individualization of the segments. 
The ripe segments are almost square in form, with the genital appa- 
ratus opening in the median line. The fully developed segments 
measure 2.5 to 4.5 mm. in length by 8 to 14 mm. in breadth. The 
total number of segments may far exceed 3000. The frequent 
occurrence of imperfect and abortive types of twin segments may be 
considered an almost distinctive feature of the bothriocephalus family 
(Wilson). The uterus presents 4 to 6 convolutions on each side, which 
become especially distinct when the segments are placed in water 
or are exposed to the air. A rosette-like appearance is then noted, 
which is quite characteristic (Fig. 83). The rosette deepens in color in 
proportion to the number of ova which the uterus contains, and toward 
the tail of the parasite, from the segments of which many or all the 
eggs have been discharged, the rosette tends to become light in color, 
and may indeed appear whiter than the surrounding parenchyma. 
The eggs (Fig. 89) are oval, 0.06 to 0.07 mm. long and about 0.045 
mm. broad ; they are enclosed in a brown envelope, at the anterior end 
of which a little lid can be recognized. Their contents consist of pro- 
toplasmic spherules, all of about the same size, which are lighter in 
color in the centre than at the periphery. In infected individuals 
they are constantly found in the stools. 

The larvae have been found in various fresh-water fishes, such as 
the perch, the ling, the turbot, in various members of the trout 
family, but they are most commonly encountered in the pike. It is 
thus readily understood why the parasite is most common in lake 
regions, as in Switzerland, northern Russia, southern Scandinavia, 
and northern Italy. It is seldom seen in middle Germany, but is 
so common in Ireland that Cobbold named it the Irish tapeworm. 
Outside of Europe it is most common in Japan. In the United States 
a few imported cases have been observed by Walker and Leidy, 
Packard, Hageestam, Riesman, Stengel, McFarland, and Wilson. 

Multiple infection has been repeatedly observed. Bottcher notes 
a case in which 100 worms were found; Roux and Eichhorst both 
speak of cases with 90, Heller of one with 38, and in Wilson's case 
2 were undoubtedly present. When more than 1 occurs the growth 
of the individual is impeded, and small specimens are then usually 
seen (three to five feet or more). Clinically the parasite is of especial 
interest, as its presence in a certain percentage of cases is associated 
with the clinical picture of a pernicious anemia; in others, how- 
ever, no deleterious effect upon the red corpuscles is noted, although 
several worms may be present in the intestinal tract. 



302 



THE FECES 



Besides in man, the worm has been encountered in the dog, cat, 
the seal, and in some water birds. The ovum, after being discharged 
in the feces, during a variable period of incubation in the water 
develops into the onchosphera, a ciliated larva with six hooklets (Fig. 
91 ) . The larva is then liberated from the ovum by passing through the 
lidded end, and by means of its cilia moves rapidly through the water. 
If not eaten by fish, it dies; otherwise it develops into the bothrio- 
cephalus measle, the plerocercoid (Fig. 90), which has both head and 
tail. Infection of man then occurs when such fish are eaten either 
raw or but partly cooked. In man the cysticercus stage has not been 
observed. 1 

Krabbea grandis, Blanchard. — This parasite has been observed in 
only one instance — in Japan. It is said to resemble certain bothrio- 
cephali which are found in seals. The genital organs are double in 
each segment. The vulva and uterus open ventrally. The worm 
attains a length of 10 m. with a breadth of 2 cm. 




Fig. 89. 



Fig. 90. 




Figs. 89 and 90. 
cerccrd. 



-Eggs and plero- 
(Braun.) 



Fig. 91. — Embryo with cilia and hooklets of 
Bothriocephalus latus. (Leuckart and Braun.) 



Trematodes. — The various forms of distoma which belong to this 
order are essentially hepatic parasites, and rarely occur in the feces. 

Distoma hepaticum, Abildgaard: syn., Fasciola hepatica (Linne) 
(Fig. 92). This, the most common liver fluke, is 28 mm. long and 
12 mm. broad; it is formed like a leaf. The leaf is provided with 
a sucker, and a second sucker may be found at its ventral surface. 
Between the two the genital opening is located, leading into a skein- 
shaped uterus. The eggs are oval, measuring 0.13 mm. in length 

1 Schaumann, Zur Kenntniss d. sogenannten Bothriocephalus-Anaemie, Berlin, 

1894. Schaumann u. Tallqvist, " Ueber d. blutkorperchenauflosenden Eigenschaf- 
ten d. breiten Bandwurms," Deutsch. med. Woch., 1898, p. 312. Runeberg, 
Deutsch. Arch. f. klin. Med., 1887, vol. xli, p. 304. Askanazy, Zeit. f. klin. Med., 

1895, vol. xxvii, p. 492. R. N. Wilson, " Bothriocephalus, Report of a. Case oi 
Double Infection," Amer. Jour Med. Sci., 1902, vol. cxxiv, p 262, 



ANIMAL PARASITOLOGY OF THE FECES 



303 



and 0.08 mm. in breadth, the anterior end being provided with a lid; 
their color is brown. In the United States the organism is practi- 
cally unknown, while in Germany it is most common in sheep. In 
the human being it is rare in both countries. It occurs in cattle, 
sheep, swine, cats, rabbits, etc. Infection occurs through a small 




Ms 




Cp 




V. sc 



Fig. 92. — Distoma hepaticum, with male Fig. 93. — Dicrocoelium (Distoma) laneeo- 

and , female genital apparatus. (From latum, Stil. and Hass; V. s., ventral sucker^ 
Ziegler, after Leuckart.) Cp, pouch of cirrus; I, intestinal furcations; 

V. sc,, vitelline sacs; T, testicles; O, ovarium; 
Ms, oval sucker; Ut, uterus. 

snail, the Linnaeus minutus, which is found, in Germany especially, 
upon watercress. 1 

Distoma lanceolatum, Mehlis, has been found in only five cases, 
all of which occurred in Germany (Fig. 93). It is much smaller 
than Distoma hepaticum, measuring 8 to 9 mm. in length, by 2 to 

1 C. W. Stiles, Jour. Comp. Med. and Vet. Arch., 1894, vol. xv, and 1895, vol. 
xvi. Huber, Trematoden. Bibliog. d. klin. Helminthol., Heft 7 u. 8, p. 283. 



304 THE FECES 

3.3 mm. in breadth. It is lancet-shaped, tapering toward the head 
end, but otherwise closely resembles Distoma hepaticum. The ova 
are 0.04 mm. long, 0.03 mm. broad, and contain fully developed 
embryos. In cattle, sheep, and hogs the organism is quite common. 1 

Distoma Buski, Lankester: syn., Distoma rhatonisii (Poirier); 
Distoma cranum (Busk); Fasciolopsis Buski (Lankester.) The 
parasite has been observed in China, Sumatra, the Straits Settle- 
ments, Assam, and India. An imported case has been described 
in the United States (Moore). It is the largest distoma occurring in 
man, measuring over an inch in length. It probably inhabits the upper 
portion of the intestine and may give rise to attacks of recurring 
diarrhea and other signs of intestinal irritation. Infection probably 
occurs through certain fishes and oysters, with certain snails as 
intermediary hosts. 2 

Distoma sibiricum, Winigradoff: syn., Distoma felinum (Rivolta). 
This parasite was found in Tomsk, by Winigradoff, in eight autop- 
sies out of one hundred and twenty-four. Askanazy also reports 
two cases of infection from eastern Prussia, in which the eggs were 
found in the stools. In one of the cases, which came to section, 
more than one hundred organisms were found in the biliary passages. 
Its length may reach 13 mm. The ova are 0.026 to 0.038 mm. long 
and 0.010 to 0.022 mm. broad. The intestine is simple and extends 
to the posterior extremity of the body. Its surface is smooth. 3 

Distoma spatulatum, Leuckart: syn., Distoma sinense (Cobbold); 
Distoma endemicum (Balz); Distoma japonicum (Blanchard). It 
has been observed in India, Mauritius, Corea, Formosa, China, 
Tonkin, and Japan, and it appears that in the two last-named coun- 
tries it is quite common. It inhabits the biliary passages and gall- 
bladder. It is distinctly pathogenic. The ova may be found in the 
stools. The parasite possibly also occurs in cats. The intermediary 
host is not definitely known; it may be some fresh-water mollusk. 
It is about 11.75 mm. long and 2 to 2.75 mm. broad. The living 
parasite is of a reddish color and translucent, so that it is possible to 
distinguish all its interior organs. The ova measure 0.028 to 0.030 
mm. in length by 0.016 to 0.017 mm. in breadth, and are enclosed in 
a colorless envelope. 4 

Other parasites belonging to this order are Distoma conjunctum 
(Cobbold), Distoma heterophyes (v. Siebold), and Amphistomum 
hominis (Lewis and McConnell). The last named appears to be 
common in elephants and has been encountered in natives of Assam, 
in two Indians in Calcutta, and in an East Indian immigrant in : 

1 Leuckart, loc. cit., p. 137. 

2 Poirier, Centralbl. f. Bakt. u. Parasit., 1888, vol. ii, p. 186. 

3 Winigradoff, cited by Braun, Centralbl. f. Bakt. u. Parasit., 1894. vol. xv, p. 
602. 

4 Blanchard, loc. cit. 



ANIMAL PARASITOLOGY OF THE FECES 



305 



British Guiana. It is quite small, measuring from 5 to 8 mm. in 
length by 3 to 4 mm. in breadth and is characterized by the large 
size of its posterior suckers. 

Distoma heterophyes is the smallest distoma, so far as we know, 
which is found in man. It occurs in Egypt and is thought to be 
innocuous. (Fig. 94.) 

Distoma conjunctum was discovered in an East Indian. Its surface 
is covered with minute spicules. It is not of much pathological 
importance. (Fig. 95.) 



L_ 



Ct. g 




Ys 



V. sc 






V. 




Fig. 94. — Cotylogonimus (Distoma) heterophyes. X 53 (v. Sieb.); C. g, cerebral ganglion; 
/, intestinal branches; Ct. g, cuticular glands; V. sc, vitelline sacs; G. c, genital cup; T, testes, 
the excretory bladder between them; L. c, Laurer's canal; R. s, receptaculum seminis, with 
the ovarium in front of it; Ut, uterus; Vs, vesicula seminalis. On the left side above, an 
egg X 700 is depicted, and below it three chitinous rodlets from the genital cup. X 700. 
(Looss.) 

Distoma haematobium and Distoma pulmonale are described in the 
sections on the Blood and the Sputum, respectively. 

Annelides. — The annelides are very common intestinal parasites, 
and of these especially the nematodes. 

Ascaris lumbricoides, Linne (Fig. 96), is the cylindrically shaped 
worm so commonly seen in children and in the insane. The head 
consists of three projections or lips, which are provided with suckers 
20 



306 



THE FECES 



Ph 




Ms 



Vsc 



V se 



T 
Z 

Ex 



Fig. 95. — Distoma conjunctum, 
Cobb [nee Lewis and Crum; nee McCon- 
nell;, from Cants f virus (Cobbold): 
Vs, ventral sucker; /.intestine; Vsc, 
vitelline sacs; Ex, excretory bladder; 
T, testes; O, ovary; Ms, oral sucker; 
Ph, pharynx; Ut, uterus. 





Fig. 97. — Ascaris mystax. (v. Jaksch. 
a, male; b, female; c, head; d, egg. 



Fig. 96. — Ascaris lumbricoides: A, 
female; B, male; C, egg; at a the female 
genital opening; c, the male spicules; b, 
the enlarged cephalic extremity, with its 
three lips. (After Perlo, from Ziegler.) 



ANIMAL PARASITOLOGY OF THE FECES 307 

and fine teeth. The male measures about 215 mm., the female about 
400 mm. in length. The tail end of the male is rolled up on its 
ventral surface like a hook, and is provided with papillae. The gen- 
ital aperture of the female is situated directly behind the anterior 
third of the body. The eggs are yellowish brown in color, almost 
round, and measure 0.06 mm. by 0.07 mm. in size; they are sur- 
rounded by an irregular albuminous envelope, which is covered by 
a tough shell; the contents are coarsely granular. 

Ascaris lumbricoides is found in all countries, and also infests the 
pig and the ox. Its presence may occasion severe nervous symptoms. 1 

Ascaris mystax, Zeder: syn., Ascaris marginata (Rudolphi); Ascaris 
alata (Bellingham) (Fig. 97). This worm is smaller and thinner than 
Ascaris lumbricoides, but otherwise very similar. The head is pointed 
and provided with wing-like projections which constitute the main 
point of difference between the two. The male measures 45 to 60 
mm. in length, the female 110 to 120 mm. Its ova are round, larger 
than those of Ascaris lumbricoides, and enclosed in a membrane 
which is covered with numerous small depressions. The worm is 
common in dogs and cats, but very rare in man. 2 

Ascaris maritima, Leuckart, also belongs to this class. It has been 
observed in only one case — in Greenland. 

Ascaris Texana (Smith-Goeth). 3 A supposedly new species, which 
has been found in a single instance in Texas. The male has not yet 
been described. 

Oxyuris vermicularis, Bremser: syn., Ascaris vermicularis (Linne); 
Ascaris grsecorum (Pallas) (Figs. 98, 99, and 100). The male is 4 
mm., the female 10 mm. long. At the head three lip-like projections 
with- lateral cuticular thickenings may be seen. The tail of the 
male is provided with six pairs of papilla? and the female with two 
uteri. The eggs are 0.05 by 0.02 to 0.03 mm. in size, and covered 
with a membrane showing a double or triple contour; in the interior, 
which is coarsely granular, the embryos are contained. 

The female worm lives in the cecum, but after impregnation 
travels downward to the rectum. Here it causes most annoying 
symptoms, which are especially distressing at night, when the organ- 
ism emerges from the anus. In doubtful cases of pruritus ani et 
vulva? an examination of the feces should be made for this parasite. 
The ova themselves do not occur in the feces. 4 

Uncinaria duodenalis (Roilliet), Ankylostomum duodenale (Dubini) : 
syn., Ankylostoma duodenale (Dubini); Strongylus quadridentatus 

1 Lutz, Centralbl. f. Bakt. u. Parasit., 1888, vol. iii, pp. 553, 584, 616. Hogg, 
Brit. Med. Jour., 1888, p. 121. Kartulis, Centralbl. f. Bakt. u. Parasit., vol. 1. 
p. 65. 

2 K. A. Rudolphi, Arch. f. Zool. u. Zoot., 1803, vol. iii, pt. ii, p. 1. Idem, Ento. 
zoorum s. verrnium intestinal, historia naturalis, Amsteraedami, ii, 2. 

3 Jour. Amer. Med. Assoc, Aug. 20, 1904, p. 542. 

4 Lutz, loc. cit. 



308 



THE FECES 



(v. Siebold), Dochmius ankylostomum (Molin); Sclerastoma duo- 
denale (Cobbold) ; Strongylus duodenalis (Schneider) ; Dochmius duo- 
denale (Leuckart) (Figs. 101 to 103). This organism belongs to 
the family Strongyloides, and is one of the most dangerous parasites 
met with in the human being. It has been found in Italy, Germany, 




a b 





1 2 

Fig. 99. — 1. Oxyuris vermicularis: a, male; 
^ilil 6, female; natural size. 2. Magnified. 




Fig. 9^. — Oxyuris vermicularis: a, sexually 
mature female; b, female filled with eggs; 
c, male. Magnification, 10. (After Heller, 
from Ziegler.) 



Fio. 100. — Eggs of Oxyuris vermicularis in 
various stages of development: a, b, c, division 
of the yolk; d, tadpole-like embryo; e, worm- 
shaped embryo. Magnification, 250. (After 
Zenker and Heller, from Ziegler.) 



Switzerland, Belgium, Egypt, and the West Indies. C. W. Stiles 
has shown that a distinct species of the hookworm exists in the United 
States as also in the West Indies, viz., in Cuba and Porto Rico, the 
Uncinaria Americana, and that in the sand regions of the South 
infection with this parasite is common. Infection occurs very largely 
through the skin and perhaps altogether so. C. A. Smith insists 
that uncinariasis exists in all cases in which ground itch has occurred 



ANIMAL PARASITOLOGY OF THE FECES 



309 





Fig. 102. — Ankylostoma duodenale, male and female. 
Natural size. (From Mosler.) 




Fig. lUo. — Head of Ankylostoma duodenale: a, buccal 
capsule; b, teeth of capsule; c, teeth of dorsal margin; 
d, oral cavity; e, ventral prominence; /, muscle layer; 
g, dorsal groove; h, esophagus. (After Schulthess, ffom 
Ziegler.) 



Fig. 101. — Male Ankylostoma duodenale: a, head; b, esophagus; c, gut; d, anal glands; 
e, cervical glands; /, skin; g, muscular layer; h. excretory pore; i, trilobed bursa; k, ribs of 
bursa: I, seminal duct; m, vesicula seminahs; ??, ductus ejaculatorius; o, its groove; p, penis; 
q, penile sheath. Magnification, 20. (After Schulthess, from Ziegler.) 



310 THE FECES 

within eight years, and that the disease is rarely if ever present in 
those who have not had ground itch within that time. 

From a pathological standpoint the parasite is of special interest, 
as its presence may give rise to severe and fatal anemia. Grie- 
singer was the first to point out that the so-called Egyptian chlorosis 
is produced by this organism. Subsequently it was shown that the 
same parasite was responsible for the anemia which developed 
among the workers on the St. Gothard tunnel, and which is com- 
mon among the brickmakers in certain districts in Germany. In 
this country the anemia of the dirt-eaters has long been known in 
the South, and has been generally attributed to the peculiar habit. 
Its real cause is now manifest. In Porto Rico the disease was very 
common until very recently and responsible for much of the severe 
anemia which was so frequent among the natives. In Germany, 
France, and Belgium the mining districts have become extensively 
infested and the eradication of the disease a serious problem. 

Outside of man the parasite is not uncommon in dogs, cattle, and 
sheep. 

The male is 6 to 11.5 mm. long, the female 10 to 18 mm. The 
head, which tapers somewhat, is turned toward the back; the mouth 
capsule is hollowed out and surrounded by 4 teeth; 1 the tail of the 
male forms a 3-lobed bursa, while that of the female tapers coni- 
cally; the genital opening is behind the middle of the body. Its 
eggs have an oval form and a smooth surface, measuring from 0.05 
to 0.06 by 0.03 to 0.04 mm. In their interior two or three segmenting 
bodies are found, which rapidly develop outside of the human body, 
so that after twenty-four to forty-eight hours embryos may be found 
in the same feces in which the eggs were observed, or fully developed 
ova may be found after allowing the feces to stand for only a few 
hours (Plate XV). When allowed to dry, the young parasites 
become encysted, but after remaining so even for from one to two 
weeks they are capable of infection. A second host for its cycle of 
development is, according to Leichtenstern, not necessary. 2 

The habitat of the adult worm is the jejunum. It is rarely found 
in the feces. Its eggs, however, are common, and should be looked 
for in every case of anemia the cause of which is not manifest, especially 
in miners, tunnel- workers, brickmakers, dirt-eaters, etc. Any speci- 
men of fecal material will answer as a rule, but it is best to procure 

1 The American species has only one dorsal, conical tooth, which projects pro- 
minently into the buccal cavity (Stiles). 

2 Centralbl. f. klin. Med., 1885, vol. vi, p. 195; Deutsch. med. Woch., 1885, 
vol. xi; 1886, vol. xii; 1887, vol. xiii. Lutz, Volkmann's Sammlung, 1885, Nos. 
255 and 256. American cases: C. W. Stiles, "The Significance of the Recent 
American Cases of Hookworm Disease," Eighteenth Annual Report of Bureau of 
Animal Industry, 1901. H. F. Harris, Amer. Med., Nov. 15, 1902, p. 776. A. J. 
Smith, Am. Jour. Med. Sci., 1903, vol. cxxvi, p. 768. C. F. Craig, ibid., p. 798; 
C. A. Smith, Jour. Amer. Med. Assoc, Aug. 27, 1904. 



ANIMAL PARASITOLOGY OF THE FECES 



111 



a thin stool, as after a purge. It is then merely necessary to mount 
a small drop on a slide and to examine the covered specimen with a 
low power; a Bausch & Lomb f is quite sufficient. A mental picture 
of the size of the eggs should be made, for I have known it to occur 
that an observer saw the eggs, but did not recognize them as such. 
Once seen, thev are easilv recognized again. 

To hatch the eggs artificially Smith recommends to mix the fecal 
materal with a small amount of soil in a Petri dish, using a sufficient 
amount of water for the purpose. There should be just sufficient 
moisture to keep the soil damp. If there is too much the cover is 
left off for an hour or so. Every two to three days a few drops of 
water are added to replenish the moisture. Under favorable condi- 
tions in this respect all the eggs will hatch within twenty-four hours; 
otherwise several days will elapse. In such cultures the larvae will 
remain alive for three or four months and can be observed with a § 
in the inverted dish. 

Trichocephalus ho minis, Schwank: syn., Trichocephalus dispar 
(Rudolphi); mastigodes (Zeder); trichuris (Buttner). This parasite, 
which belongs to the family Tricho- 
trachelides, is formed like a whip, 
the last end being the head end, 
while the tail end is very much 
thicker. The male measures 46 
mm. and the female 50 mm. in 
length. The eggs are brownish in 
color, measuring 0.05 by 0.06 mm. 
in size, and present a doubly con- 
toured shell, with a depression at 
each end, closed by a lid. The 
contents are coarsely granular. 
The organism is said to be the 
most widely distributed intestinal 
parasite, occurring in Europe, 
North America, Asia, Africa, and 
Australia. Its habitat is the cecum. 
The living worm is only rarely 
found in the feces 1 (Fig. 104). 

Trichina spiralis, Owen, is rarely 
found in the feces. The male 
measures 1.5 mm. in length, and is provided with four papillae 
between the conical lips. The female is 3 mm. long. The 
uterus is situated nearer the head than the ovary, which opens into 
it. Fertilization occurs in the intestinal canal. The eggs develop 
into embryos in the uterus, emerge from this, and penetrate the 




-Trichocephalus trichiuris. On 
the left, male; on the right, female with the 
anterior extremity embedded in the mucous 
membrane of the'intestine. Below, egg. 



1 Ermi, Berlin, klin. Woch., 1886, vol. xxiii, p. 614. 



312 



THE FECES 



intestinal walls, whence they are carried by the blood current to the 
muscles. The diagnosis of sporadic cases has been greatly facili- 
tated by the discovery of Brown that eosinophilia, often of high 
grade, is practically of constant occurrence during the acute stage 
of the disease. In doubtful cases a small piece of muscle tissue 
(biceps, gastrocnemius) may be excised and examined for young 






- mm 










Fig. 105.— Trichina spiralis in muscle. 



trichinas. With the naked eye the cysts appear as minute little white 
specks. The worms can be rendered easily visible by placing a bit 
of the tissue in glycerin containing 5 per cent, of acetic acid; after a 
few minutes it is pressed out between two slides and examined with 
a low power (Fig. 105). While it is believed that trichinosis is less 
common in the United States than in Germany, there can be no 
doubt that it is not nearly as rare as was believed. Many light cases 
go practically unrecognized. 

Strongyloides intestinalis (Bavay): syn., Anguillula intestinalis 
(Bavay); Anguillula stercoralis (Bavay); Rhabditis stercoralis 



ANIMAL PARASITOLOGY OF THE FECES 313 

(Bavay); Leptodera stercoralis (Bavay, Cobbold), Leptodera intes- 
tinalis (Bavay, Cobbold); Strongyloides intestinalis (Bavay, Grassi); 
Pseudorhabditis stercoralis (Bavay, Perroncito) ; Rhabdonema, 
strongyloides (Leuckart); Rhabdonema intestinale (Bavay, Blan- 
chard). 

In the feces of patients infested with the parasite in question the 
eggs of the mother-worm are only rarely found, and the adult worm 
itself probably never appears unless an anthelmintic has been admin- 
istered and active catharsis established. Instead we find embryos 
(rhabditic form) measuring about 0.33 by 0.022 mm. in size. If the 
stools are kept, uncovered, at a temperature of about 37° C, their 
larvae undergo development and reach full growth and sexual dif- 
ferentiation in almost five days. The length of the full-grown 
female is about 1 mm.; its breadth about 0.04 mm. The body is 
cylindrical, slightly diminishing in size anteriorly and tapering to 
a sharp point posteriorly. When the worm retracts forcibly, slight 
transverse furrows may be seen. The mouth possesses dis- 
tinct lips and is continuous with a triangular esophagus, which 
beyond a constriction dilates again into a second ovoid enlarge- 
ment. The intestine which follows ends in a little protrusion on 
one side of the body near the base of the tail. A little below the 
middle of the body, and on the ventral side, is the vulva, which leads 
to the uterus, extending from the intestinal ventricle to a point near 
the anus. Here the eggs may be massed in varying numbers. Some- 
times the young have actually broken the shell of their eggs and may 
be seen free in the uterus; but more commonly the ova, on deposition, 
contain well-formed motile embryos (filariform brood). The male 
is about one-fifth smaller than the female. The testicle ends at the 
base of the tail, in two small, horn-like spicules with tapering ends, 
which are curved inward. These spicules contain canals; they are 
of equal size and situated symmetrically on a transverse plan. The 
tail is coiled in the same direction as the spicules, and is half as long 
as that of the female. 

The sexually mature and differentiated forms just described repre- 
sent the Anguillula stercoralis of Bavay. They represent an inter- 
mediate generation, developing outside of the body, which forms a 
link in the chain of development of the mother-worm, the Anguil- 
lula intestinalis (Leuckart). 

Ordinarily infection takes place through the larvse of the sexually 
differentiated form. These filariform embryos are longer than the 
rhabditiform brood of Anguillula intestinalis (Fig. 106). They are 
provided with a cylindrical esophagus descending down to about 
the middle of the body, and a tail, which, instead of terminating in a 
fine point, is apparently truncated at its extremity. On maturation 
they give rise to the Anguillula intestinalis, which is encountered 
throughout the upper gastro-intestinal tract, especially in the lower 



314 



THE FECES 



part of the duodenum and the upper part of the jejunum, though 
occasionally they have also been found throughout the entire jeju- 
num and in the upper part of the ileum. On several occasions they 
have been found in the stomach. 

Anguillula intestinalis, viz., the parasitic mother- worm, is, accord- 
ing to Rovelli, parthenogenetic, while Leuckart expressed the 






Fig. 106. — Strongyloides embryo (rhabditiform variety). The stool contained many red cells. 



opinion that it might be hermaphroditic. Its length is about 2.20 
mm., and its average breadth 0.03 mm. The body tapers a little 
anteriorly, and terminates posteriorly in a conical tail, the extremity of 
which is appreciably rounded and even a trifle dilated. The mouth 



ANIMAL PARASITOLOGY OF THE FECES 315 

is without horny armature, and shows three small lips. It opens 
into a cylindrical esophagus, which occupies about one-fourth of 
the length of the animal, and shows neither swellings nor striations. 
The intestine extends nearly to the posterior extremity of the body, 
but is almost invisible in the middle part owing to the presence of 
a large, elongated ovary. The vulva is situated in the posterior 
third of the animal, and the uterus contains usually five or six rather 
elongated ova. The anus is situated toward the base of the tail. 




Fig. 107. — A, egg of Strongyloides intestinalis (parasitic mother-worm); B, rhabditiform 
embryo; C, filariform embryo, derived by direct transformation from a rhabditiform 
embryo. (Taken from Thayer.) 

The eggs are of a yellowish-green color, rather opaque, and appar- 
ently finely granular (Bavay); in their general appearance they 
resemble those of the uncinaria (Fig. 107). 

While infection originally takes place through the filariform larvae 
of Anguillula stercoralis, an auto-infection with the larvae may also 
occur without the intervention of the sexually differentiated forms, 
by a direct transformation from the rhabditiform embryos of the 
parasitic mother-animal, and there is evidence to show that this 



316 THE FECES 

latter cycle is indeed more common There is no evidence to show 
that the sexually mature intermediate generation ever develops in 
the intestinal tract during life. 

The time elapsing between infection with the filariform larvse 
and the appearance of rhabditiform embryos in the stools is about 
seventeen days. 

The parasite is the recognized cause of the so-called Cochin-China 
diarrhea, and is of further interest from its resemblance to Anky- 
lostoma duodenale, with which it is not infrequently found asso- 
ciated. Excepting in very rare instances, it does not cause intestinal 
ulceration, and it is supposed that the injurious effects of the para- 
site are purely mechanical. It is possible, however, that these may 
also be owing to the irritating action of its excretory products. The 
clinical manifestations of the disease are mainly those of a chronic 
diarrhea and a comparatively mild anemia. There are usually three 
or four pasty stools a day. 

The organism was first discovered in individuals who had con- 
tracted severe diarrhea in Cochin-China. Grassi and Parona later 
found the worm in Italy, and at the building of the St. Gothard 
tunnel it was frequently seen in association with the ankylostoma. 
Thayer was the first to find it in the United States, and it is interest- 
ing to note that two of his three cases must have become infected in 
either Maryland or Virginia. The third case may have originated 
in Austria; in it the anguillula was associated with amebas and the 
Trichomonas intestinalis ; it ended fatally, being complicated by liver 
abscess. Since then additional cases have been reported in the 
United States by Moore, Price, Lamar, and others. 

Other cases have been observed in Belgium, Holland, Martinique, 
Brazil, Sicily, the Dutch Indies, Egypt, Germany, Spain, and the 
Philippine Islands. 

Literature. — Grassi, Centralbl. f. Bakt. u. Parasit., 1887, vol. ii, p. 413. Leich- 
tenstern, Deutsch. med. Woch., 1898, p. 118. Perroncito, Arch. p. 1. sci. med., 
1881, No. 2. Compt.-rend. de l'Acad. des sci., 1882, No. 1. Teissier, ibid., vol. 
cxxi, p. 171. Bavav, ibid., 1876, vol. lxxxiii, p. 694; ibid., 1877, vol. lxxxiv, p. 
266. Normand, ibid., 1876, p. 316. W. S. Thayer, Jour, of Exper. Med., 1901, 
vol. vi, No. 1 (full literature to 1901). M. L. Price, Jour. Amer. Med. Assoc, 
Sept. 12, 1903 (literature to date since Thayer's paper). 



Chemistry of the Feces. 

Reaction. — The reaction of the feces is normally usually alkaline, 
sometimes neutral, rarely acid, the alkalinity being due to ammoniacal 
fermentation, the acidity to lactic and butyric acid fermentation. 

In disease also the reaction of the stools is variable and of but little 
clinical interest. In typhoid fever an alkaline reaction is so constantly 
met with that this symptom might possibly be of value in doubtful 



] 



ANIMAL PARASITOLOGY OF THE FECES 31 7 

cases. It may, however, also be neutral, amphoteric, or even acid. 
In acute infantile diarrhea an acid reaction is the rule, but exceptions 
also are not infrequent. Normal stools of sucklings are acid, the 
degree of acidity, according to Langstein, corresponding to about 
2.1 to 3.7 per cent, of normal NaOH for 100 grams of the moist feces. 
General Composition. — The following table, taken from Gautier, 
will give an idea of the composition of fresh feces, calculated for 
1000 parts by weight: 

Adult man. Suckling. 

Water 733.00 851.3 

Solids 267.00 148.7 

Total organic material 208.75 137. I 1 

Total mineral material 10. 95 2 13.6 

Alimentary residue 83 . 00 

The organic material yielded: 

Aqueous extract 53 . 40 53 . 50 

Alcoholic extract 41.65 8.20 

Ethereal extract 30.70 17.60 3 

In addition, there are gases, which vary in quantity according to 
the nature of the food ingested, such articles as beans, bread, pota- 
toes, etc., increasing the amount very considerably. 

Milk diet. Meat diet. Vegetable diet. 
Per cent. Per cent. Per cent. 

Carbon dioxide 9-16 8-13 21-34 

Hydrogen 43-54 0.7-3 1.5-4 

Marsh gas . . *. . . . 0.09 26-37 44-55 

Nitrogen 36-38 45-64 10-19 

Of these gases, carbon dioxide is partly referable to alcoholic and 
butyric acid fermentation, and partly to albuminous putrefaction. 
Marsh gas is formed during the fermentation of cellulose, while the 
nitrogen has partly been swallowed and is partly referable to albu- 
minous putrefaction. A portion also is probably derived from the 
blood, and it may be mentioned in this connection that the enormous 
quantities of carbon dioxide so often discharged in cases of hysteria 
are undoubtedly referable to this source, the gas passing from the 
blood through the gastro-intestinal mucous membrane into the stomach 
and intestines. 

In order to give a general idea of the chemical constituents of 
the feces these may be divided into: 

1. Food material which could be assimilated, such as starches, 
fats, and a small amount of non-assimilated albuminous material. 

2. Indigestible substances, such as chlorophyll, gums, pectic 
products, resins, various coloring matters, nucleins, chitin, and 
insoluble salts, viz., silicates, sulphates, earthy phosphates, ammonio- 
magnesium phosphate, etc. 

1 Including 54 parts of mucin, epithelium, and calcareous salts. 

2 Not comprising earthy phosphates. 

3 Of this 3.2 is cholesterin. 



318 THE FECES 

3. Products derived from the digestive canal, as mucus, partly 
transformed biliary acids, dyslysin, cholesterin, lecithin. 

4. Substances in process of absorption, as emulsified fats, fatty 
acids, leucin, and biliary acids. 

5. Products of decomposition, referable to microbic activity, such 
as fatty acids, comprising the entire series from acetic to palmitic 
acid, the latter being especially abundant; lactic acid, phenol, cresol, 
indol, skatol, excretin, leucin, and tyrosin; phenyl-propionic, phenyl- 
acetic, hydroparacumaric, and parahydroxyl-phenyl-acetic acids; 
ammonium carbonate, and ammonium sulphide. 

6. Products of metabolism eliminated through the intestines; urea, 
uric acid, and xanthin bases. 

7. Pigments: stercobilin, hematin, hydrobiiirubin, coloring matter 
derived from the blood, and, in abnormal conditions, bile pigments. 

8. Water. 

9. Gases, as carbon dioxide, marsh gas, hydrogen, and nitrogen. 
It is impossible to give here a detailed description of the various 

chemical constituents which have been mentioned. Only the most 
important ones, and those especially interesting from a physiological 
and pathological standpoint, will be considered. 

Phenol, Indol, and Skatol. — Phenol, indol, and skatol are formed 
during the process of albuminous putrefaction, and are constant con- 
stituents of the feces. A small portion is absorbed from the intestinal 
canal, and appears in the urine in combination with sulphuric acid 
and to a slight extent also with glucuronic acid. Previously, however, 
the indol and skatol are oxidized to indoxyl and skatoxyl, respectively 
(see Urine). 

To demonstrate the presence of phenol, indol, and skatol in the 
feces, we may proceed as follows: 

The feces are diluted with water, acidified with phosphoric acid, 
and distilled. Volatile fatty acids, together with phenol, indol, 
and skatol, pass over. The distillate is neutralized with sodium 
carbonate and again distilled. During this process phenol, indol, 
and skatol pass over, the fatty acids remaining behind as sodium 
salts. In order to separate phenol from indol and skatol, the distil- 
late is alkalinized with potassium hydrate and again distilled. The 
phenol now remains behind, and may be obtained in pure form by 
distilling with sulphuric acid; in this final distillate its presence may 
be demonstrated by the following reactions: 
•1. With ferric chloride phenol yields an amethyst-blue color. 

2. With bromine-water a crystalline precipitate of tribromophenol 
is obtained. 

3. Treated withMillon's reagent — i. e., the acid mercuric nitrate — 
a red color develops. 

Indol and skatol pass over after treating the above mixture of 
the three with potassium hydrate and distilling. These two bodies 



ANIMAL PARASITOLOGY OF THE FECES 319 

may then be separated from each other by taking advantage of their 
different degrees of solubility in water. 1 

Indol forms small plates, melting at 52° C, which are easily soluble 
in hot water, alcohol, and ether; its odor is feculent. 

Reactions of indol: (1) When treated with nitric acid and a little 
sodium nitrite a crystalline red precipitate of the nitrate of nitroso- 
indol is obtained. (2) A small piece of pine wood moistened with 
an alcoholic solution of indol acidified with hydrochloric acid is 
colored a cherry red. (3) A small amount of an aqueous solution of 
indol is shaken with a few drops of Ehrlich's dimethyl-amino-ben- 
zaldehyde solution (which see). A cherry-red color develops either at 
once or upon the application of heat. 

Skatol crystallizes in plates which melt at 95° C. They are soluble 
with more difficulty in water than indol, and emit a feculent odor. 

Reactions of skatol: (1) With nitric acid and sodium nitrite only 
a milky cloudiness results. (2) Pure skatol does not color pine wood 
moistened with hydrochloric acid; but if a bit of the wood is satu- 
rated with a dilute alcoholic solution of skatol and then immersed 
in strong hydrochloric acid, it assumes a cherry-red and later a 
bluish-violet color. (3) With nitric acid of a specific gravity of 1.2 
it gives a marked xanthoproteic reaction on boiling — i. e., a yellow 
color which turns to orange upon the addition of an excess of ammonia. 

Whenever there is increased intestinal putrefaction the fatty acids, 
phenol, indol, and skatol, will, of course, be found in increased 
amounts. 2 

Fatty Acids. — The fatty acids which may be found in the feces 
are the following: 



Formic acid . 


. H.COOH 


= C H 2 2 


Acetic acid 


. CH 3 .COOH 


= C 2 H 4 2 


Propionic acid 


. CH 3 .CH 2 .COOH 


= C 2 H 6 2 


Butyric acid . 


. CH,.(CH 2 ),.COOH 


= C 4 H 8 2 


Isobutyric acid . 


. (ch 3 ) 2 .ch:cooh 


= C 4 H 8 2 


Valerianic acid . 


. CH 3 .(CH 2 ) 3 .COOH 


= C 5 H 10 O 2 


Caproic acid . 


. CH 3 .(CH 2 ) 4 .COOH 


= C 6 H 12 2 


Capric acid . '. 
Palmitic acid 


. CH 3 .(CH 2 ) 8 .COOH 


= C 10 ri 20 C) 2 


. CH 3 .(CH 2 ) M .COOH 


= C 16 xi 32 (J 2 


Stearic acid . 


. CH 3 .(CH 2 ) 16 .COOH 


= C 18 H 36 2 



These acids are derived partly from fats, partly from carbohydrates, 
and to some extent also from proteins. 

Cholesterin. — Cholesterin (C 26 H 44 0) occurs in small amounts in 
almost all animal fluids. It is found also in various tissues of the 
body, especially in the brain. Its origin and mode of formation in 
the various organs of the body, as well as the cause of its presence 
in the alimentary canal, are as yet unknown. It crystallizes in 
colorless, transparent plates, the margins and angles of which usually 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co. 

2 Ibid 



320 THE FECES 

present a ragged appearance. (See Fig. 75, page 272.) It is prac- 
tically insoluble in water, dilute acids, and alkalies. In boiling 
alcohol it is readily soluble and crystallizes out from this solution on 
cooling; it is likewise easily soluble in ether, chloroform, and benzol. 

Tests for cholesterin: 1. Under the microscope add a drop of 
concentrated sulphuric acid to some of the crystals; they gradually 
disappear, the edges assuming a yellowish-red color. 

2. Dissolve a few crystals in chloroform, add concentrated sul- 
phuric acid, and shake the mixture: the chloroform assumes a blood- 
red to a purplish-red color, while the sulphuric acid at the same time 
shows marked fluorescence. 

The Biliary Acids.— The biliary acids found in the feces are: 
glycocholic acid (C 26 H 43 N0 6 ), taurocholic acid (C 26 H 45 NS0 7 ), and 
cholalic acid (C 24 H 40 O 5 ). 

The two former occur normally in the bile, and can be decomposed 
into cholalic acid and glycocoll, and cholalic acid and taurin, respect- 
ively ; as this process of decomposition takes place ordinarily in the intes- 
tines, the third acid — i. e., cholalic acid — is always found in the feces. 

In order to demonstrate the biliary acids, the fatty acids, phenols, 
indol, and skatol are first removed by distillation with phosphoric 
acid. The residue is taken up with water and boiled, and the filtered 
liquid precipitated with lead acetate and a little ammonium hydrate. 
The biliary salts of lead are contained in the precipitate, from which 
they can be removed by washing with water and finally boiling the 
precipitate with alcohol. The washings are filtered and the lead salts 
transformed into sodium salts by treating the filtrates with sodium 
carbonate. After further filtration the filtrate is evaporated to dry- 
ness and the residue extracted without alcohol. Upon evapora- 
tion the salts of the acids sometimes crystallize out as such, while 
more often a dirty amorphous precipitate is obtained, which may be 
rendered crystalline by treating with ether. The amorphous residue, 
however, can be employed for making the necessary tests. 

Pettenkofer's Test. — A small amount of the substance is dissolved 
in water and treated with two-thirds its volume of concentrated 
sulphuric acid, care being taken that the temperature does not exceed 
60° or 70° C. While stirring, a 10 per cent, solution of cane sugar 
is added drop by drop. If biliary acids are present, the solution 
assumes a beautiful red color, which on standing turns a bluish violet. 
This test depends upon the action of furfurol, derived from the sul- 
phuric acid and cane sugar, upon the biliary acids. 

Pigments. — The principal pigment of normal feces is termed 
stercobilin, and was first isolated from this source by Vanlair and 
Masius. 1 Owing to its great similarity to hydrobilirubin, it has even 
been regarded as identical with it, but Garrod and Hopkins 2 have 

1 Centralbl. f. d. med. Wiss., 1871, vol. ix, p. 369. 

2 On Urobilin, Jour, of Physiol., 1898, vol. xxii, p. 451. 



ANIMAL PARASITOLOGY OF THE FECES 321 

conclusively shown that whereas the urobilin of the urine and the 
stercobilin of the feces are identical in composition, as also in 
properties, they differ conspicuously from hydrobilirubin, and espe- 
cially in the much smaller percentage of nitrogen which they con- 
tain, viz., 4.11, as compared with 9.22 per cent. It is derived from 
bilirubin, and formed in the upper regions of the large intestine 
more especially, as the result of bacterial activity. 1 This explains 
the observations that as a rule the meconium and the solid excreta 
of the first day or two of life contain no urobilin, and that the pigment 
also disappears, when for any reason the bile is prevented from 
entering the intestinal canal. 

To isolate the pigment from the feces, the material is first extracted 
with alcohol. The alcoholic extract is evaporated to dryness, the 
residue is extracted with water, the aqueous solution acidified with 
sulphuric acid and saturated with ammonium sulphate, when on 
shaking with chloroform or a mixture of chloroform and ether the 
pigment is taken up by the organic solvent. 

The free pigment is a brown, amorphous substance of a character- 
istic odor, and melts at. a temperature below 100° C. On cooling, 
it forms a brittle, shellac-like material, which is said to be quite char- 
acteristic. It is soluble in ether, chloroform, water, and amyl alcohol. 
On treating its solutions with zinc chloride and ammonia a beautiful 
green fluorescence is obtained. Such solutions then show three bands 
of absorption, of which the one between C and F is the most char- 
acteristic. (See also Urinary Urobilin.) 

Test for stercobilin : A small amount of feces is stirred up in water 
and a few c.c. of the resultant mixture treated with an equal amount 
of a saturated aqueous solution of bichloride of mercury. A normal 
stool, owing to the presence of stercobilin, then turns a pinkish red, 
which is the more marked the fresher the material. A green color is 
abnormal and denotes the presence of bile pigment. 

Bile Pigment is normally absent from the feces. It occurs in 
large amounts in catarrhal conditions of the small intestine, and may 
be demonstrated by Gmelin's method, viz., a drop of the filtered 
liquid, or a particle of the colored fecal matter, is brought into con- 
tact with a drop of fuming nitric acid, when the yellow color will 
be seen to pass through the various shades of the spectrum, the 
green shade being the most characteristic. At times, however, it is 
not possible to obtain a positive reaction in this manner, although 
bile pigment is present. In such cases the examination should be 
conducted under the microscope, and attention directed to bile-stained 
epithelial cells, leukocytes, particles of mucus, and crystals. 

1 A. Schmidt, Verhandl. d. XIII. Congresses f . inn. Med., 1895, p. 320. Vaughan 
Harley, Brit. Med. Jour., 1896, vol. ii, p. 898. Macfadyen, Nencki, and Sieber, 
Arch. f. exper. Path. u. Pharmakol., 1891, vol. xxviii, p. 311. 
21 



322 THE FECES 

Hematoporphyrin, to judge from the investigations of Stokvis 1 
and Garrod, 2 is likewise a normal component of the feces, but occurs 
only in traces. Garrod states that with Saillet's 3 method, the basis 
of which is extraction with acetic ether, after the addition of acetic 
acid, he invariably found traces, comparable with those which nor- 
mally are present in the urine. He also states that he found con- 
siderably larger amounts of the pigment in the meconium, both in 
that expelled during the first day or two of life, and in that removed 
from the intestines of stillborn infants. 

The presence of these normal traces has been referred by some to 
the^ingestedjblood-coloring matter of red meat and vegetable chloro- 
phyll.^ Garrod, however, finds that the hematoporphyrin does not 
disappear when these articles of diet are withdrawn, and while 
admitting that the ingested hemoglobin and chlorophyll may possi- 
bly be converted, in part at least, into hematoporphyrin, he concludes 
that the greater portion is derived from endogenic sources. On the 
whole, the evidence seems now in favor of the view that the hemato- 
porphyrin which is found both in the urine and in the feces originates 
within the liver, and is eliminated into the intestinal canal in the 
bile. (See also Hematoporphyrinuria.) 

Purin Bodies. — The purin bases of the feces are derived from the 
nuclei of desquamated epithelial cells, from the nucleoproteids of 
bacteria and leukocytes, from the secretions of the intestinal glands 
and the pancreas, and from the ingested food. The normal quantity 
according to Schittenhelm 4 varies between 0.1109 and 0.1669 purin 
nitrogen. When excessive amounts of meat, thymus gland, or guanin 
are added to the diet a large proportion of the purin nitrogen is elimi- 
nated in the feces in the next twenty-four hours. In diarrhea the 
fecal purins are increased. 

Guanin, adenin, xanthin, and hypoxanthin are all represented, the 
first two prevailing. 

Mucin. — According to Hoppe-Seyler, mucin is a constant con- 
stituent of the feces, both under physiological and pathological 
conditions. Normally, however, it is never possible to recognize its 
presence either with the naked eye or with the microscope. A satis- 
fying test for the rapid demonstration of mucin in the feces does not 
exist. The old test of Hoppe-Seyler indicates nucleo-albumin, but 
not true mucin. To this end the feces are digested with water and 
treated with an equal volume of milk of lime; the mixture is allowed 
to stand for several hours, when it is filtered and the filtrate tested 
with acetic acid. In the presence of nucleo-albumin a cloud develops 
upon addition of the acid. 

1 Nederl. Natuur-en Geneeskundig Congres, 1899, p. 378. 

2 "The Urinary Pigments in their Pathological Aspects," Lancet, Nov. 10, 1900. 

3 Rev. de med., 1896, vol. xvi, p. 542. 

4 Zeit. f. physiol. Chem. 1903, vol. xxxix, p, 1.99. Walker Hall, Jour. Pathol, 
and Bact., March, 1904, p, 246, 



ANIMAL PARASITOLOGY OF THE FECES 323 

Albumin is demonstrated in the feces by treating repeatedly with 
water slightly acidified with acetic acid. The filtrate is then examined 
for albumin according to methods given elsewhere (see Urine). Under 
normal conditions these reactions prove negative. Pathologically, serum 
albumin has been observed in cases of typhoid fever and chlorosis. 

Albumoses are normally absent from the feces. They have been 
observed in typhoid fever, dysentery, tuberculous ulceration, purulent 
peritonitis with perforation into the gut, atrophic cirrhosis, and carci- 
noma of the liver. Acholic stools are also usually rich in peptones. 

The albumoses are demonstrated in the following manner: the 
feces are digested with water, so as to form a thin mush; they are 
then boiled, filtered while hot, and the filtrate examined for albumin, 
so as to be sure that all of this has been removed. The mucin is 
removed by treating with lead acetate, when the filtrate is examined 
for albumoses as described in the chapter on Gastric Contents. 

Carbohydrates. — Of the carbohydrates, starch, glucose, and certain 
gums may be found. In order to demonstrate these the feces are 
boiled with water, filtered, and evaporated to a small volume. This 
solution may now be tested with phenylhydrazin or Trommer's 
reagent for glucose (see Urine), and with a solution of iodopotassic 
iodide for starch (see Saliva). 

In normal breast-fed infants sugar is only demonstrable in traces 
in the stools. Langstein 1 finds that the presence of more than traces 
of glucose in the stools of milk-fed infants may be regarded as a 
diagnostic symptom of a catarrhal process in the duodenum. 

Ptomains. — Of ptomains, only two have been isolated from the 
feces, viz., putrescin and cadaverin. They have been found in Asiatic 
cholera, in cholerina, dysentery, and in connection with cystinuria. 
In cholera and cystinuria their amount may be quite large. Baumann 
and v. Udranszky obtained 0.5 gram of the benzoylated compounds 
from the collected feces of twenty-four hours. Such findings are 
exceptional, however; more often the result is negative or traces only 
are found; such has been my own experience and that of others. 
(See Ptomains in the Urine.) In cholera the cadaverin seems to pre- 
dominate, while in cystinuria more putrescin is found. 

To isolate the diamins in question, the feces are digested with 
alcohol which has been acidified with sulphuric acid. The alcoholic 
extract is evaporated, the residue dissolved in water, and further 
benzoylated, as described in the section on Urine. 

MECONIUM. 

By meconium are meant those masses which are first excreted from 
the bowel after birth. It is a thick, tenacious, greenish-brown mate- 

1 Jahresb, f. Kinderheilk., vol. vi, Heft 3, 



324 THE FECES 

rial which has accumulated during the intra-uterine life of the infant. 
Microscopically, a few cylindrical epithelial cells, a few fat droplets, 
numerous cholesterin crystals, bilirubin crystals, and lanugo hairs 
are found. 

Microorganisms are absent, but soon after suckling has com- 
menced they appear in abundance. The most important of those 
which are then constantly present are the Bacillus lactis aerogenes, 
which predominates in the small intestine, and the Bacillus coli 
communis, which is found more particularly in the large intestine. 
Both have already been described. In addition to these, the Proteus 
vulgaris, Streptococcus coli brevis, Micrococcus ovalis, tetragencoccus, 
Saccharomyces cerevisise, Saccharomyces rubra, and a few less impor- 
tant microorganisms have been found. 

Chemically, meconium contains bilirubin in considerable amount 
(recognizable by Gmelin's reaction), biliary acids, fatty acids, chlo- 
rides, sulphates, phosphates of the alkalies, and their earths. It 
does not contain urobilin, glycogen, albumoses, lactic acid, tyrosin, 
or leucin. 

An idea may be formed of its composition from the following 
analysis of Zweifel: 1 

Water 79 . 8-80 . 5 per cent. 

Solids 19.5-20.2 

Mineral matter 0.978 

Cholesterin 0.797 

Fats 0.772 

1 Hellstrom, Arch. f. Gynak., 1901, vol. lxiii, Heft 3. 






CHAPTEE V. 
THE NASAL SECRETION. 

In the nasal secretion, which normally is small in amount, trans- 
parent, colorless, odorless, tenacious, and of a slightly saline taste, 
pavement-epithelial cells in large numbers, ciliated epithelial cells, as 
well as some leukocytes and an enormous number of microorganisms, 
are found. Its reaction is alkaline. 

In acute coryza the amount is diminished at first, but soon a very 
copious secretion occurs, which contains numerous epithelial cells 
and microorganisms. When complicated with an ulcerative condi- 
tion pus is observed in considerable amount. 

Occasionally, as in cases of traumatism, cerebral tumors, etc., 
cerebrospinal fluid is discharged through the nose, and may be 
recognized by the fact that it is free from albumin and contains a 
substance which reduces Fehling's solution. 

Of pathogenic organisms, the tubercle bacillus and the bacillus of 
glanders may occur in ulcerative diseases of the nose, their presence 
indicating the existence of the corresponding affection. In ozena a 
large diplococcus has been described by Lowenberg, which is said to 
be characteristic of the disease. Oidium albicans has been observed 
in rare cases. The Meningococcus intracellularis of Weichselbaum, 
which is now regarded as the cause of epidemic cerebrospinal 
meningitis, has also been demonstrated in the nasal secretion of 
healthy individuals. In ordinary cases of coryza the Micrococcus 
catarrhalis is frequently found. 

Ascarides and other entozoa have also been found. 

Charcot- Leyden crystals have been observed in the nasal secretion 
in cases of bronchial asthma and in connection with nasal polpyi. 
Their presence is usually accompanied by the simultaneous occur- 
rence of large numbers of eosinophilic leukocytes. 

Literature. — Reimann, Baumgarten's Jahresber., 1888, vol. iii, p. 417, 
Lowenberg, Deutsch. med. Woch., 1885, vol. xi, p. 6, and 1886, vol. xii, p. 446. 
Tost, ibid., p. 161. Gerber u. Podack, Deutsch. Arch. f. klin. Med., 1895, vol. 
liv, p. 262. Levden, Deutsch. med. Woch., 1891, vol. xvii, p. 1085. Sticker, 
Zeit. f. klin. Med., 1888, vol, xiv, p. 81. Nothnagel, Wien. med. Blatter, 1888, 
Nos. 6, 7, 8. 



CHAPTEE VI. 

THE SPUTUM. 
GENERAL TECHNIQUE. 

The sputum should be collected in receptacles so constructed as 
to permit of their complete and easy disinfection. The paper spit- 
cups which are figured in the accompanying illustrations (Pigs. 108 
and 109) are admirably adapted for this purpose, as they may he 
destroyed immediately after use. 

When working with sputa which are known or suspected to be of 
tuberculous origin, the greatest care should be exercised to keep the expec- 
toration from drying and becoming disseminated in the air. Negligence 
in this respect may residt in the most serious consequences. 

The macroscopic examination of sputa is most conveniently 
carried out by placing small portions of the material upon a plate of 





Sanitary spit-cups. 



Fig. 109. 



ordinary window-glass, of suitable size, which has been painted black 
upon its lower surface, and covering the same with a second, smaller 
plate. If it is desired to examine individual constituents which 
have been discovered in this manner, the upper plate is slid off until 
the particle in question is uncovered, when it may be removed to a 
microscopic slide and examined under a higher power. 

It is also very convenient to have a portion of the laboratory 
table painted black, when unstained plates of glass may be utilized. 
If these measure about 15 by 15 cm. and 10 by 10 cm., respectively, 
fairly large quantities of sputa may be examined in situ with a low 
power. 



GENERAL CHARACTERISTICS OF SPUTA 327 



GENERAL CHARACTERISTICS OF SPUTA. 

Amount. — The amount of sputum expectorated in the twenty- 
four hours varies within wide limits, depending largely upon the 
nature of the disease. Thus, only a few cubic centimeters may 
be eliminated, or the amount may reach 600 to 1000 c.c, and even 
more. Very large quantities are expectorated in cases of pulmonary 
hemorrhage and edema of the lungs, sometimes following thoracen- 
tesis, also following perforation of accumulations of pus from the 
thoracic or abdominal cavities into the respiratory passages; further- 
more, in cases in which large vomicae of tuberculous or gangrenous 
origin exist, and finally in cases of abscess of the lung, bronchiectasis, 
and even in simple bronchial blennorrhea. In incipient phthisis, 
acute bronchitis, and in the first and second stages of pneumonia, 
on the other hand, the amount is usually small. 

In private practice, as well as in hospital work, an idea should 
always be formed of the amount expectorated in the twenty-four 
hours, especially in cases in which this is abundant. It is apparent 
that a copious and long-continued expectoration cannot continue 
without exerting very detrimental effects upon the patient's general 
nutrition; in cases of pulmonary phthisis, for example, Renk has 
shown that 3.8 per cent, of all nitrogen eliminated in such cases is 
removed in this manner. Lenz in his experiments found even 5 
per cent. 

Consistence. — The consistence of the sputum corresponds, in a 
general way at least, to its amount, and may vary from a liquid to 
a highly tenacious state. The cause of the tenacity of the sputum 
is but imperfectly understood. Mucin does not appear to be the 
most important factor, as this occurs in diminished amount in pneu- 
monic sputa, which are noted for their high degree of tenacity. 
Kossel 1 has suggested that the phenomenon may be due to the pres- 
ence of nucleins or nuclein derivatives, while others refer it to the 
presence of abnormal albuminous bodies of unknown character. 
However this may be, sputa are not infrequently seen where it is 
possible to invert the cup without losing a drop of its contents. This 
is observed especially in cases of acute croupous pneumonia up to the 
time of the crisis, providing that a catarrh of the bronchi does not 
exist at the same time. It is noted, furthermore, immediately after 
an attack of acute bronchial asthma, and also in the intial stage of 
acute bronchitis. In cases of edema of the lungs, on the other hand, 
the sputa are liquid and present the general characteristics of blood 
serum, being covered, like all albuminous liquids when brought into 
contact with the air, by a frothy surface layer. The sputa observed 

1 Zeit. f. klin. Med, 1888, vol. xiii, p. 152. 



328 THE SPUTUM 

in cases of acute pulmonary gangrene, pulmonary abscess, putrid 
bronchitis, and following perforation into the lungs of an empyema 
or an accumulation of pus situated beneath the diaphragm, are fluid 
and consist of pure pus. 

Color. — The color of the sputa may vary greatly. They may be 
perfectly clear and transparent, gray, yellow, green, red, brown, and 
even black. Purely mucoid expectoration is almost transparent and 
colorless, as is also the sputum of pulmonary edema when not mixed 
with blood or pus. 

The larger the number of leukocytes the more opaque does the 
sputum become, assuming at first a white, then a yellow, and finally 
a greenish color, the latter being usually indicative of the presence 
of pus. The green color, however, may be due to other causes. 
Green sputa may thus be observed when bile pigment has become 
admixed with the sputa, as in cases of liver abscess perforating into 
the lung, or in cases of jaundice, and especially in pneumonia during 
lysis, in pneumonia ending in abscess, and in subacute, caseous pneu- 
monia. The same is seen in pulmonary chloroma and may also 
occur in pulmonary carcinoma. In cases of amebic liver abscess 
with perforation into the lung the sputa usually present a color 
resembling anchovy sauce, which is very characteristic. 1 

The inhalation of particles of carbon gives the sputum a grayish 
or even a black color; the same or an ochre-yellow or red color is 
observed in cases of siderosis due to oxide of iron. Blue sputa are 
seen in workers with blue dyes (methylene blue, ultramarine), etc. 

A red color is usually indicative of the presence of blood, the shade 
depending upon the character of the disease. It is seen especially 
after the formation of cavities, in caseous pneumonia, in incipient 
phthisis, heart disease, etc. The shade will further depend upon the 
length of time that the blood, no matter what its origin may be, has 
remained in the lungs. In pulmonary gangrene a dirty, brownish- 
red color is observed, owing to the presence of methemoglobin, and, 
to some extent also, of hematin. Quite characteristic is the chocolate 
color which is observed when a croupous pneumonia terminates in 
necrosis and gangrene. Equally characteristic is the rusty and prune- 
colored expectoration seen in ordinary cases of pneumonia. Occasion- 
ally a breadcrust brown is observed in cases of gangrene and abscess 
of the lung, the color being due to the presence of hematoidin or 
bilirubin. A light-brown color may be seen in cases of chronic passive 
congestiou, as in mitral disease. 

Odor. — Most sputa are odorless. Under certain conditions, how- 
ever, there may be a marked odor. In cases of pulmonary gangrene 
or putrid bronchitis the stench is frightful. A somewhat similar, 
slightly sweetish odor is observed in certain cases in which putre- 

1 See C. E. Simon, Johns Hopkins Hosp. Bull., November, 1890. 






GENERAL CHARACTERISTICS OF SPUTA 329 

factive organisms have entered the lungs, and there exert their action 
upon the accumulated sputa, in the absence of gangrene, as in cases 
of bronchiectasis, perforating empyema, and where ulcerative proc- 
esses are taking place in the lungs, whether these be of tuberculous 
origin or not. An odor like that of old cheese is occasionally observed 
in cases of perforating empyema; under such conditions tyrosin is 
usually found. This body, however, has nothing to do with the odor 
of the sputa; both factors are merely indicative of certain putre- 
factive changes going on in the lungs. 

Specific Gravity. — The specific gravity of sputa varies within wide 
limits; mucous sputa have a specific gravity of 1.004 to 1.008, purulent 
sputa one of 1.015 to 1.026, and serous sputa one of 1.037 or more. 

Configuration of Sputa. — As a general rule, the following forms 
of sputa, which may be termed pure sputa, present a homogeneous 
appearance : 

Mucoid sputa, 

Purulent sputa, j. Homogeneous sputa> 

Serous sputa, | to r ' 

Sanguinous sputa, J 

with one exception, perhaps — the typically rusty sputa of croupous 
pneumonia; while mixtures of any two or three of these may be 
classed as heterogeneous sputa: 

Mucopurulent sputa, 

Mucoserous sputa, j. Hete eous ta> 

Serosangumous sputa, j fe ^ 

Sanguino-mucopurulent sputa J 

The so-called sputum crudum of the first stage of acute bronchitis 
may be regarded as an example of a purely mucoid sputum. A 
purely purulent sputum is usually indicative of the perforation of an 
empyema or any other accumulation of pus into the lungs or bronchi, 
of pulmonary abscess, or of bronchial blennorrhea. A purely serous 
sputum is found in cases of pulmonary edema, and a purely hemor- 
rhagic sputum in cases of pulmonary hemorrhage. 

Of the heterogeneous sputa, the most important are the so-called 
nummular sputa of the second and third stages of phthisis. These 
are characterized by the fact that when thrown or expectorated into 
water they sink to the bottom, and there form coin-like disks, from 
which property they have received there name. Such sputa are 
mucopurulent in character, and contain a focus of almost pure pus 
embedded in a more or less homogeneous mass of mucus. Quite 
different from these are the so-called sputa globosa, which consist 
of fairly dense, roundish, grayish- white masses; they are secreted in 
old cavities which have become lined with a granulation membrane. 

Occasionally, as in putrid bronchitis, bronchorrhea, bronchiectasis, 
and gangrene of the lungs, exquisite sedimentation is observed. Such 
sputa when collected in a conical glass present three distinct zones: 



330 THE SPUTUM 

the one at the bottom contains the cellular elements, the second the 
pus serum; the third or superficial layer consists of mucus and con- 
tains many air bubbles. From this long shreds of sedimentous 
material sometimes hang down. 



MACROSCOPIC CONSTITUENTS OF SPUTA. 

Cheesy Particles. — The presence of small, cheesy particles, which 
are occasionally found at the bottom of the spit cup is sometimes very 
important. They vary in size from that of a millet-seed to that of a 
pea, and are observed especially in the second and third stages of 
phthisis. Usually they contain tubercle bacilli in large numbers, 
and frequently also elastic tissue. Not to be confounded with these 
are small, caseous masses which are at times expectorated by perfectly 
normal individuals, and also by patients suffering from acute tonsil- 
litis, ozena, etc., and which in part come from the tonsils or mucous 
cysts (Dittrich's plugs); others may be derived from the bronchi. 
Formerly they were regarded as tubercles, and in hypochondriac 
individuals their expectoration may cause a great deal of anxiety. 
As a rule, they are expectorated unaccompanied by pus or even mucus; 
rubbed between the fingers they emit an extremely offensive odor, 
which is referable to the presence of fatty acids; microscopically they 
consist of bacteria, fatty acid crystals, fat globules, and cellular 
detritus. 

Elastic 1 issue. — In cases in which active parenchymatous destruc- 
tion of the lungs is going on bits of elastic tissue may be found which 
are visible with the naked eye. The search is facilitated by spreading 
out the sputum between two plates of glass, upon a dark background, 
and searching with a hand lens. In tuberculosis the particles are 
quite small, while in abscess and gangrene they may attain the size 
of a pea. Their macroscopic demonstration should be followed 
by a careful microscopic examination (which see). 

Particles of cartilage from tuberculous ulcers of the larynx, trachi, 
and bronchi are less common, as is also the occurrence of tumor frag- 
ments. 

Fibrinous Casts. — Fibrinous casts are observed in croupous pneu- 
monia, immediately before or after resolution has taken place, as 
also in fibrinous bronchitis (Fig. 110), and in diphtheria when the 
membrane has extended into the finer ramifications of the bronchi. 
These casts may vary in size from 15 cm. in length by several milli- 
meters in thickness to fragments which measure only from 0.5 to 3 
cm. in length. The casts observed in pneumonia, usually from the 
third to the seventh day, are of the latter size or even smaller, being 
derived from the ultimate twigs of the finest bronchioles. Those 
found in fibrinous bronchitis stand between these two in size, being 






MACROSCOPIC CONSTITUENTS OF SPUTA 331 

casts of smaller and medium-sized bronchi. Attention is usually 
attracted to the presence of such casts by their white color; often, 
however, they are yellowish brown or reddish yellow, owing to the 
presence of blood-coloring matter; at other times they are enveloped 
in mucus, when their recognition may become quite difficult. Such 
casts are fairly firm; they branch dichotomously, usually six to ten 
times. The larger branches contain a lumen, while the smallest twigs 
are solid. Microscopically they consist of a large number of fibers, 




Fio. 110. — Expectorated east from a case of fibrinous bronchitis. Three-fourths natura 
size. Drawn from fresh specimen. (After Bettmann.) 

which are arranged longitudinally or in a net-like manner, and con- 
tain blood corpuscles and epithelial cells in their meshes. When 
treated with Weigert's fibrin-stain, they are sometimes beautifully 
resolved; at other times the fibrin reaction is not nearly so marked as 
one would expect. The individual casts consist of a variable number 
of lamina arranged concentrically, those contained in the centre being 
much folded and involuted. Most of the branches are cylindrical; 
some of the larger ones are flat. Charcot-Leyden crystals have at 
times been observed in these formations. 



332 THE SPUTUM 

Small casts composed of the mycelium of fungi have also been 
described. 

Whenever it is desired to examine sputa for casts, it is best to pick 
out particles that look promising, upon a dark surface, and then to 
shake them out in water. 

Literature. — M. Bettmann, Amer. Jour. Med. Sci., 1902, vol. cxxiii, p. 304 (a 
full review of all cases in the literature up to 1902 is here given). Devillers and 
Renon, La presse medicale, 1899. 

Curschmann's Spirals. 1 — Quite distinct from the formations just 
described are the so-called spirals of Curschmann, which are observed 
especially in cases of true bronchial asthma, but occur also in acute 
and chronic bronchitis, in croupous pneumonia and in chronic phthisis, 
though to a far less extent. Upon careful examination they will be 
seen to consist of thick, yellowish-white masses, which exhibit a 
spirally twisted appearance, and are characterized, moreover, by 
their more solid consistence and light color. Microscopically they 









/'.mm 



ilk 




Fig. 111.— A Curschmann spiral from a case of true bronchial asthma. (Enlarged.) 

are composed of a spirally twisted network of extremely delicate 
fibrils, containing epithelial cells and numerous leukocytes; the latter 
are almost all of the eosinophilic variety. 2 Usually, but not invariably, 
Charcot-Leyden crystals also are seen. 3 The spirally twisted mass is 
found to be wound around a central, very light and clear thread, 
which usually has a zigzag course (Fig. 111). 

Other formations, probably mere varieties of those just described, 
have also been observed, in which the central thread is absent or in 
which the spiral arrangement is deficient. The spiral form, how- 
ever, with the central thread, must be considered as the most char- 
acteristic. Their length and breadth may vary a great deal, but 
rarely exceed 1 to 1.5 cm. Their occurrence seems always to indi- 

1 Levden, Virchow's Archiv, 1872, vol. liv, p. 328. Curschmann, Deutsch 
Arch. f. klin. Med., 1883, vol. xxxii, p. 1, and vol. xxxvi, p. 578. v. Jaksch, 
Centralbl. f. klin. Med., 1883, vol. iv, p. 497. 

2 Schmidt, Zeit. f. klin. Med., 1892, vol. xx, p. 92. v. Noorden, ibid., p. 98. 

3 Leyden, loc. cit. 



MACROSCOPIC CONSTITUENTS OF SPUTA 333 

cate a desquamative catarrh of the bronchi and alveoli, but practi- 
cally nothing is known concerning their formation. If in a given 
case the diagnosis rests between true bronchial and what may be 
termed reflex asthma, the presence of these formations points to the 
existence of the former disease. Chemically, the spirally wound 
mass seems to consist of a mucinous substance, while the central 
thread is possibly of fibrinous origin. 

Charcot-Leyden crystals (Fig. 112), which are usually absent at 
the beginning of an attack of asthma, at which time only the spirals 
are observed, may develop from the spirals when these are kept for 
several days. They will be considered later in studying the chemistry 
of the sputum. 

Echinococcus Membranes. — Echinococcus membranes may come 
from a perforating cyst of the liver, kidney, or lung. They consti- 
tute rather thick, and at the same time tough, pieces of membrane 
(Fig. 113); occasionally entire sacs are seen, of the color of white 





^) Fig. 113.— Wall of a hydatid cyst, 

showing the laminated structure; not 
Fig. 112. — Charcot-Leyden crystals. (Scheube.) magnified. (Davaine.) 

porcelain, in sections of which it is possible to make out a fibril- 
lated structure. (See also Animal Parasites in the Sputum.) 

Concretions. — The expectoration of concretions which have been 
formed in dilated portions of the bronchi or in tuberculous cavities, 
or of calcified bronchial glands that have found their way into the 
lungs is rare. Curious examples of the occurrence of such concre- 
tions have been reported. Andral cites a cases of phthisis in which 
within eight months 200 stones were expectorated, and Portal men- 
tions a case in which 500 were thus expelled. 1 

Foreign Bodies . — Foreign bodies which have accidentally entered 
the air passages and have remained there for a long time may also 
be found in the sputum. Heyfelder mentions a case in which a man 
coughed up a wooden cigar-holder with pus and blood after eleven 
and a half years. 

1 L. W. Atlee, " Bronchial Concretions," Amer. Jour. Med. Sci., 1901, vol. cxxii, 
p. 49. Fiessinger, " Calcule pulmonale," Jour, de med., 1902, No. 29, 



334 THE SPUTUM 



MICROSCOPIC EXAMINATION OF THE SPUTUM. 

Under this heading it is necessary to consider leukocytes, red 
blood corpuscles, epithelial cells, elastic fibers, corpora amylacea, 
parasites, and crystals. 

Leukocytes. — Leukocytes, usually polynuclear in character, are 
found in every sputum in considerable numbers, embedded in a 
homogeneous, more or less tenacious material. At times they con- 
tain fat droplets, or granules of pigment, such as carbon or hematoidin. 
Their number varies considerably, being naturally greatest in cases 
of perforating abscess, empyema, putrid bronchitis, etc. 

While the leukocytes which usually are found in the sputum are 
of the neutrophilic variety, eosinophiles may also be observed, and 
especially in asthmatic sputa, in which they predominate. Free 
eosinophilic granules are then also seen, and I have repeatedly 
observed specimens in which the spirals (see above) were literally 
covered with these granules (Plate XVII). The presence of eosino- 
philic leukocytes is, however, not characteristic of the sputa of 
bronchial asthma, as they may be met with in other diseases as 
well. Teichmuller has pointed out that they are present in a large 
percentage of tuberculous cases, and may be found months before 
tubercle bacilli can be demonstrated. He regards their occurrence 
as evidence of a defensive struggle on the part of the body, which 
is most evident in fairly strong individuals. In recovery a gradual 
increase in their number is noticeable, and a diminution, Teich- 
muller thinks, is indicative of a relapse, or, if the diminution occurs 
rapidly, of florid consumption. These statements, however, lack 
confirmation and are probably too dogmatic. Ott, Fuchs, Bett- 
mann, Turban, and Cohn, in fact, deny the prognostic significance 
of the eosinophilic cells in cases of phthisis; and Cohn states, as 
the result of an examination of 100 cases, many of which were com- 
paratively early, that the occurrence of eosinophilic leukocytes is 
fairly uncommon in tuberculous sputa. Stadelmann 1 also states that 
he has been unable to verify Teichmuller's observations. On the 
other hand, he has been able to confirm the observation which has 
been repeatedly made, that large numbers of eosinophilic cells appear 
in the sputum following hemoptysis. Teichmuller has also described 
an "eosinophilic" bronchitis, which is said to differ from other forms 
of the disease in the abundance of eosinophilic cells which are encoun- 
tered. The sputum in such cases is described as transparent, mucoid, 
and loose, with yellow, purulent admixtures. It is said to be mark- 
edly different from the tough, thick sputa of bronchial asthma, 

1 Discussion on tuberculosis, Deutsch, med, Woch,, 1901, vol. v, p. 210. 



> 



PLATE XVI. 



'.& 


















Sputum from Case of Bronchial Asthma, showing Large Numbers 
of Eosinophilic Leukocytes and Free Granules. 

It will be noted that the leukocytes are all mononuclear. (Eye-piece 1, objective 1-8, Bausch & Lomb.) 



MICROSCOPIC EXAMINATION OF SPUTUM 335 

Typical spirals are absent, but rudimentary forms may be encountered. 
Charcot-Leyden crystals are present. 1 

Very curiously the majority of the eosinophilic cells which are met 
with in the sputum (notably in asthma) are mononuclear; they are 
not myelocytes, however, but probably mononuclear histogenetic 
forms. 

Griinwald 2 states that in the sputa of the most diverse diseases 
cells are met with which contain a hypoeosinophilic granulation, and 
that the granules in question may also occur outside of the cells in 
the absence of evidence of special cell destruction. These gran- 
ules, in contradistinction to the true eosinophilic cells, lose their 
color on treating with an acid, and readily take up the blue stain on 
subsequent staining with methylene blue. Griinwald states, how- 
ever, that a sharp line of distinction does not exist between the two 
varieties of granules, and that intermediary conditions exist, as also 
transitions between oxyphilic and basophilic granules in the nature 
of an amphophilic granulation. 

To demonstrate eosinophilic leukocytes in the sputum, smears are 
made as usual, slightly fixed by drawing through the flame of a 
burner, and stained for two minutes in a 0.5 per cent, alcoholic solu- 
tion of eosin. The preparations are then immersed in 50 per cent, 
alcohol to the point of decolorization, when they are counterstained 
with methylene blue, briefly washed with water, and dried. The 
eosinophilic granules and the red cells in part hold the eosin dye. 

Basophilic leukocytes (mast-cells) have also been observed in the 
sputa. 

Red Blood Corpuscles. — The presence of red blood corpuscles in 
small numbers does not, by any means, indicate serious pulmonary or 
cardiac disease, as they may be found in almost any sputum, and 
especially in that of individuals who smoke much or live in a smoky 
atmosphere; they are, without doubt, derived from the catarrhally 
inflamed bronchial or tracheal mucosa. Whenever they occur in 
large numbers, however, their presence becomes important. They 
may be observed in acute bronchitis, pneumonia, edema of the 
lungs, bronchiectasis, abscess, gangrene — in fact, in all pulmonary 
diseases. Their occurrence is most important in phthisis, and is. in 
fact, one of the most constant symptoms of the disease. 

The form of the red corpuscles will depend upon the length of 
time that they have remained in the lungs, and all gradations from 
the typical red corpuscle to its shadow, or even fragments, may be 

1 Teichmiiller, "Die eosinophile Bronchitis," Deutsch. Arch. f. klin. Med., vol. 
lxiii, p. 444. See, also, K. Schonbrod, Ueber den gegenwartigen Stand der 
Beurtheilung der eosinophilen Zellen im Blute und im Sputum, Inaug. Diss., 
Erlangen, 1895. A. Hein, Ueber das Vorkommen eosinophiler Zellen im Sputum , 
Inaug. Diss., Erlangen, 1894. 

2 "Studien liber d. Zellen im Auswurf, etc," Virchow's Archiv. 1899, vol. clviii, 
p. 297, 



336 THE SPUTUM 

observed. In pneumonia the microscopic examination may at 
times be disappointing, the appearance of the sputum suggesting 
that red corpuscles in large numbers are present, while, as a matter 
of fact, they are almost all destroyed, the color being due to altered 
pigment. It may even be necessary to depend upon chemical methods 
to clear up the question. It should be remembered that the presence 
of blood pigment is not always indicated by a red color, but that it 
may also assume a golden-yellow or even a greenish tinge, owing to 
certain chemical changes which have taken place. The golden-yellow 
and the grass-green sputa observed in cases of pneumonia during 
convalescence belong to this class. 

To demonstrate the presence of traces of blood in the sputum, the 
aloin or guajac test (see Feces) may be employed, after first boiling 
the sputum with 20 per cent, caustic alkali solution and susequently 
neutralizing with acetic acid. 

Epithelial Cells . — Epithelial cells are found in practically every 
sputum. They are mostly of the pavement variety and may be 
derived from the mouth, pharynx, and the upper larynx. Many of 
the cells are full of invading bacteria, which may lead to their 
entire destruction. Cylindrical epithelial cells, providing they do 
not come from the nose, indicate in a general way an inflammatory 
condition of the lower larynx, trachea, or bronchi. As a rule their 
form is so much altered that it is often difficult to recognize them; 
they may thus become polyhedral, cuboidal, or even round, and can 
then hardly be distinguished from leukocytes. Actively moving cilia 
may be found only in perfectly fresh sputa, immediately after being 
expectorated, but are very rarely seen. 

Formerly much importance was attached to the so-called alveolar 
epithelial cells (Fig. 114) as an aid in diagnosis. Buhl thus regarded 
them, particularly when undergoing fatty or myelin degeneration, 
to be pathognomonic of pulmonary disease, and especially of that 
form of pneumonia which has been termed essential idiopathic 
desquamative pneumonia. Bizzozero, 1 however, as well as others, 
have shown that these cells not only occur in almost every known 
pulmonary disease, but that they are present also in the so-called 
"normal" expectoration which at times is obtained upon making 
a forcible expiration. They are round, oval, or polygonal cells 
varying in size from 20 fi to 50 //. They may contain one, two, or 
three oval nuclei, which are rather small and provided with nucleoli. 
Usually the latter are hidden beneath numerous granules. Some of 
the granules are albuminous, but most of them are either pigment 
granules, fatty granules, or myelin granules. The rriyelin granules 
were first discovered by Virchow 2 , and termed myelin granules on 

1 Microscopie clinique, 2d ed. Frarifaise, Paris, 1885. 

2 Virchow's Archiv, 1854, vol. vi, p. 562. 



PLATE XVII. 



FIG. 1 






■ 






// 



- i 






Tuberculous Sputum Stained by Gabbett's Method. The Tubercle Baeil 
are seen as Red Rods, all else is Stained Blue. (Abbott.) 



FIG. 2. 



mm 






* . m&. 



•.» *&?■■ 



% 



c % 



^Sr 






Heart Disease Cells, showing Alveolar Epithelial Cells, Loaded Down 
with Granules of Hematin. 



MICROSCOPIC EXAMINATION OF SPUTUM 



337 



account of their resemblance to mashed nerve matter. They are 
distinguished from the other forms by their clear, pale, colorless 
appearance, and the fact that at times fine concentric striations can 
be detected. These forms may be round, but more often they are 
irregular. Chemically, the myelin droplets have been shown to con- 
tain a considerable amount of protagon, besides traces of lecithin and 
cholesterin. 1 They are readily soluble in alcohol, somewhat so in 
chloroform and ether. They swell in water and stain yellow with 
iodine. They are colored but little by the anilin dyes and do not 
turn black on treating with osmic acid. 

Sometimes myelin granules are found together with fatty and pig- 
ment granules in the same cell. 

The sputa of chronic bronchitis referable to heart disease are 
characterized by the presence of so-called heart-disease cells. These 




Fig. 114. — Epithelium, leukocytes, and crystals of the sputum. (Eye-piece III, objective 
8 A, Reichert.) a, a', a", alveolar epithelium: b, myelin forms; c, ciliated epithelium; d, 
crystals of calcium carbonate; e, hematoidin Crystals and masses; f, f, f, white blood cor- 
puscles; g, red blood corpuscles; h, squamous epithelium. (,v. Jaksch.) 

are alveolar epithelial cells containing hematoidin granules (Plate 
XVIII, Fig. 2). They appear to be most numerous in cases of mitral 
disease, but may also occur in congestive affections of the broncho- 
pulmonary apparatus, even with the heart intact. 2 

Liver cells may at times be observed in the sputa in cases of liver 
abscess, and are easily recognized by their characteristic form. 

Elastic Tissue. — Much more important from a clinical stand- 
point are the elastic fibers and shreds of elastic tissue which may 
be found in sputa. They vary much in length and breadth, and 
are provided with a double, undulating contour: they are usually 
curled at their ends. Very often they exhibit an alveolar arrange- 
ment (Fig. 115), which at once determines their origin. 



1 A. Schmidt, "Ueber Herkunft u. chem. Natur d. Myelinformen d. Sputums," 
Berlin, klin. Woch., 1898, p. 73. See, also, Zoja, Maly's Jahresberichte, vol 
xxiv, p. 694. 

2 R. C. Regolo, Caz. d. Ospedali, Milano, vol. xxii, No. 135. 

22 



338 THE SPUTUM 

Whenever present, elastic tissue is an absolute indication that a 
destructive process is going on- in the" lungs. It is found in cases of 
abscess of the lungs, bronchiectasis, occasionally in pneumonia, pul- 
monary gangrene and infarct, and, most important of all, in phthisis, 
in which it is said to be present in 90 per cent, of all cases. This 
percentage, which was obtained by Dettweiler and Setzer in 1878, 
is unquestionably too high in comparison with what is seen today, 
where the diagnosis of tuberculosis is made much earlier. In gan- 
grene of the lung elastic tissue is generally said to be absent, but Osier 
states that he has never seen a case without it, and that usually it 
occurs in large fragments. 

In every case it is necessary to determine whether the elastic tissue 
has not been ntroduced from without, and it may hence be stated as 
a rule that it can only be regarded as absolutely characteristic when 
showing the alveolar arrangement. 

In order to demonstrate the presence of elastic tissue in the sputum 
the following method is very convenient: A small amount of the thick, 




Fig. 115. — Elastic fibers in the sputum. (Eye-piece III, objective 8 A, Keichert.) 

(v. Jaksch.l 



(v. Jaksch 

purulent portion of the sputum is pressed into a thin layer between 
two pieces of plain window-glass, 15 by 15 cm. and 10 by 10 cm. 
The particles of elastic tissue appear on a black background as grayish- 
yellow spots, and can be examined in situ under a low power. Or, the 
upper piece of glass is slid off till the piece of tissue is uncovered, 
when it is picked out and examined on a slide, first with a low and 
then with a higher power. At first there will be some difficulty in 
distinguishing with the naked eye between elastic fibers and particles 
of bread, or milk globules, or collections of epithelium and debris, 
but with practice such mistakes are rarely made, and the microscope 
always reveals the difference. 

If only very little elastic tissue is present, it is necessary to examine 
large quantities of sputum with a moderately low power, and best 



ANIMAL PARASITOLOGY OF SPUTUM 339 

after the addition of a solution of sodium hydrate. The sputum is 
boiled with a 10 per cent, solution of the reagent, an equal volume 
being added; the boiling is continued until a homogeneous solution 
has been obtained; after dilution with four times its volume of water 
it is allowed to settle for twenty-four hours or centrifugalized and the 
sediment examined at once. 

May 1 recommends the following method of demonstrating the 
presence of elastic tissue in sputum: The material in question is 
heated on a boiling water bath with an equal volume of a 10 per 
cent, solution of sodium hydrate until it has all apparently dissolved. 
The mixture is then centrifugalized and the supernatant fluid 
decanted. The sediment is treated with about 2 c.c. of an orcein 
solution prepared according to the formula of Unna-Tanzer, viz., 
orcein, 1 gram; absolute alcohol, 80 c.c; distilled water, 40 c.c; 
concentrated hydrochloric acid, 40 drops. On adding the stain, 
owing to the remaining alkali, the color turns violet; a few drops 
(3 to 5) of hydrochloric acid are added until the original color of the 
stain returns. The tube is then placed for from two to five minutes 
in boiling water, after which acid alcohol (concentrated hydrochloric 
acid, 5 c.c; 95 per cent, alcohol, 1000 c.c; distilled water, 250 c.c.) 
is added to decolorize. The mixture is again centrifugalized and 
the sediment washed once or twice more with the acid alcohol by 
centrifugation and decantation. The sediment is then examined 
directly, when the elastic tissue fibers may be recognized by their 
more or less intense brownish-violet color. 



ANIMAL PARASITOLOGY OF THE SPUTUM. 

Protozoa. Entamoeba Dysenterise. — In cases of amebic abscess of 
the liver with perforation into the lung the Amoeba coli may be demon- 
strated in the sputa. Such sputum commonly presents the anchovy 
sauce appearance already mentioned. As a rule the amebas are not 
numerous and slide after slide may have to be examined before a single 
organism is discovered. The material should be kept at body tem- 
perature and the slides warmed. A Bausch and Lomb \ or Leitz 
6 or 7 is used (see also Amebas in Feces). Only actively moving 
organisms are diagnostic. 

Trichomonads have at times been observed in cases of gangrene 
of the lung, and in the pus removed postmortem from lung cavities. 
They are identical with the Trichomonas vaginalis of Donne. 

Cercomonads have been found in the sputum and in the Dittrich 
plugs in gangrene of the lung. 

Cestodes. Taenia Echinococcus. — Portions of echinococcus cvsts, 
viz., pieces of membrane (Fig. 114) and hooklets (Fig. 119), are 

1 Deutsch. Arch. f. klin. Med., 1900, vol. lxviii, p. 427. 



340 



THE SPUTUM 



occasionally seen when the parasite has lodged in the lungs or in the 
neighboring organs. The disease is not common in this country. 
Lyon 1 collected 241 cases in the United States and Canada up to 
July 1, 1901. 91 per cent, occurred in foreigners. In Canada a 
large proportion is referable to the Icelandic immigrants in Manitoba. 





Fig. 117. 






& k «:j v-~% 



Fig. 116. 



Fig. 118. 



Fig. 116. — Taenia echinococcus. X 50. The cirrus pouch, the vagina, uterus, ovary, shell- 
gland and vitellogene gland and the testicular vesicles at the sides are recognizable in the 
second proglottis; the uterus partly rilled with eggs, as well as the cirrus pouch and the vagina. 

Fig. 117. — Section through an echinococcus cyst with brood capsules. 

Fig. 118. — A piece of the wall of an echinococcus veterinorum stretched out and seen from 
the internal surface. X 50. A few brood capsules with scolices directed toward the interior 
and exterior. (Thomas.) 



Thomas/ 2 of Adelaide, has thoroughly investigated the disease in 
Australia, where it is quite common. 

The adult parasite (Fig. 116), Taenia echinococcus (v. Siebold), 
is a three- or four-segmented tapeworm, 4 to 5 mm. in length, whose 



1 N. Y. State Jour. Med., Oct., 1902. 

2 Hydatid disease, 1884. 



ANIMAL PARASITOLOGY OF SPUTUM 



341 



habitat is the intestinal canal of the dog, dingo, jackal, wolf, etc. 
The larval or cystic form develops in cattle, sheep, swine, rabbits, 
etc., and is also found in man. The ova, 0.067 mm. in diameter, 
are introduced by food, water, or by inhalation in dust. In the 
digestive tract the minute embryo, freed of its resistant envelope by 
the digesting juices of the stomach, bores its way through the intes- 
tinal wall, and finds a resting place in the liver, lung, or other part 
of the body, there developing into the cystic form that may attain 
enormous size. 

The primary or mother cyst may produce daughter cysts, these latter 
granddaughter cysts, and these a third generation, often in great 
number; so that the cavity may be filled with cysts of varying size, 
formed by exogenous or endogenous growth. On the other hand, the 
single cyst may remain sterile — aceph- 
alocyst — or may produce scolices (Fig. 
117) which are attached by pedicles 
to the lining of the vesicles or brood 
capsules in which they develop. Each 
scolex, or echinococcus head, 0.4 to 
0.25 mm. in diameter, is a round or 
oval body with a head capable of 
protrusion or retraction. There is a 
single or double circlet of hooklets 
around, and four suckers behind 
the rostellum. The body is partly 
covered with calcareous particles. 
These scolices may ordinarily be 
found in hydatid-cyst contents. 

Hydatid membrane (Fig. 113) varies 
in thickness according to the size of 
the cyst, a mother-cyst membrane 
being often ■§- inch or thicker; the 
smaller cysts have walls of greater 
delicacy. It is usually pearly or grayish white, opaque, and of 
gelatinous consistency, but the thin walls of the daughter cysts may 
be perfectly clear and transparent. The membrane consists of two 
layers: (1) the ectocyst, of regular laminae of chitinous-like material, 
readily torn on manipulation, the innermost layers whiter and softer 
than the outer; (2) the delicate, soft, granular endocyst, consisting 
of a mass of delicate polygonal cells without distinct nuclei. From 
this the scolices and daughter cysts are developed. The ectocyst 
usually lies in close apposition to the fibrous adventitious capsule 
formed by the organ in which the hydatid is present. "The ectocyst, 
known also as the cubicula by Continental writers, presents under 
the microscope a peculiar stratified structure which is quite charac- 
teristic. It shows no appearance of fibers or cells, and even under 




Hooklets of echinococcus: 
a, Echinococcus veterinorum; b, Taenia 
echinococcus, three weeks after infection; 
c, adult Taenia echinococcus; d, three 
forms of hooklets outlined one within 
the other. (Leuckart.) 



342 THE SPUTUM 

high magnifying powers it exhibits a nearly hyaline or at most a 
faintly granular appearance" (Thomas). 

When a hydatid cyst of the lung, liver, or neighboring tissue has 
ruptured into the larger or smaller divisions of the bronchi, quantities 
of clear, watery fluid, giving the characteristic tests for hydatid fluid 
(see Cystic Contents), may be coughed up and be found to contain 
perhaps : 

(a) Small cysts full of clear fluid, from the size of a pin's head 
upward — the daughter or granddaughter cysts. 

(b) Whitish, dot-like bodies just visible to the naked eye when 
single, or more evident when grouped together in colonies— the 
scolices, or echinococcus heads (Fig. 118). 







Fig. 121. 






'■-\i ./— \ ' \ ;/ 

Fig. 120. Fig. 122. 

Fig. 120. — Paragoninius westermanni (Kerb.). X 10. (Leuckart.) Mouth, pharynx, intestinal 
branches; at the sides of which the vitelline sacs are observed. The genital pore is behind the 
ventral sucker, and next to it, at the left, the ovary; at the right, the uterus; the two testes at 
the back; the excretory vessel in the middle. 

Fig. 121. — Paragonimus westermanni (Kerb.) (natural size). To the left, dorsal aspect; to the 
right, ventral aspect. (Katsurada.) 

Fig. 122 — Egg of Paragonimus westermanni (Kerb.) from the sputum. X 1000. CKatsurada.) 

(c) Some of the component parts of the cysts or scolices, viz. : 

1. Collapsed cysts — the well-known "grape skins," or pieces of the 
gelatinous membrane of a mother or daughter cyst. 

2. Hooklets and calcareous corpuscles from the bodies of the 
scolices, visible only under the microscope. 

Where the hydatid has suppurated before rupture, pus in large or 
small amount takes the place of the clear fluid or is mixed with it, the 
other elements being recognized on examination. 
^Microscopic Examination of Hydatid Material. — A piece of mem- 
brane (often yellowish and shreddy in degenerating cases) is picked 



ANIMAL PARASITOLOGY OF SPUTUM 343 

up with forceps, placed on a slide, a drop or two of water applied, 
and lightly crushed under the cover-glass. At the torn edges of the 
membrane the characteristic laminated structure can be readily seen 
with the low power (Fig. 113). It does not stain readily, but staining 
is unnecessary. A section may be cut with the freezing microtome 
and stained with carmine. 

Sputa may continue to be expectorated from a hydatid cavity of 
the lung for months or years, and are then usually of a purulent or 
mucopurulent character, perhaps blood-tinged. A thick smear on a 
slide may reveal, when examined with a low power, pieces of laminated 
membrane or hooklets. A piece of membrane, if seen on floating 
the sputa in water, should be picked out with forceps. Tubercle 
bacilli are sometimes found in the sputa of cases of pulmonary hydatid. 
When a hydatid of the liver has ruptured into a bronchus the sputa 
may be bile-stained. 1 

Trematodes. Distoma Pulmonale (Lung Fluke). — A form of pul- 
monary disease closely simulating phthisis and associated with pul- 
monary hemorrhage is very common in Japan, and has been shown 
to be referable to the presence of a parasite in the lungs, Distoma 
pulmonale (Balz) — syn., Distoma westermanni (Kerbert), Distoma 
Ringeri (Cobbold), Paragonimus westermanni. The parasite is 8 
to 10 mm. long, 4 to 6 mm. wide, rounded very markedly in front, 
less so posteriorly. The color during life is a reddish brown. The two 
sucking disks are nearly equal in size. The ova are brown, with a thin 
shell and lidded. They measure from 80 to 100 fi in length and 40 to 
60 /j. in breadth. The worm and its ova are found in the sputum. 
If the sputum is shaken in water and the water renewed from time to 
time, in the course of a month or six weeks (according to the tempera- 
ture) a ciliated embryo is developed in each ovum. When the ovum is 
mature, on placing it on a slide and exercising slight pressure on the 
cover-glass, the operculum will be forced back and the embryo will 
emerge and at once begin to swim and gyrate in the water (Manson). 
Outside of Japan the parasite has been found in Corea and Formosa. 
In the United States it has been found in the cat and in the dog; in 
the human being one case, occurring in a Japanese student, has been 
reported. Many Charcot-Leyden crystals are found in the sputum 
at the same time. 

Literature. — C. D. Stiles, "Distoma Westermanni," Johns Hopkins Hosp. 
Bull., 1894, p. 57. Brown, Die thierischen Parasiten, etc., Stuber, Wurzburg, 
1895. 

Distoma Haematobium. — Manson found the ova of a species of 
Distoma haematobium in the bloody expectoration of a Chinese 
who had lived for some time on the island of Formosa, 

1 For the above account of the component parts of hydatid material I am 
indebted to my friend Dr. John Ramsay, of Launceston, Tasmania. 



344 THE SPUTUM 



BACTERIOLOGY OF THE SPUTUM. 

Tubercle Bacillus. — From macroscopic examination it is impos- 
sible to decide whether or not a particular sputum is of tuberculous 
origin. At times a sputum may have a suspicious appearance, but 
it is never possible to speak with certainty from simple inspection, 
as a mucoid sputum may contain tubercle bacilli in large numbers, 
while a mucopurulent sputum may be entirely free from them, and 
vice versa. Reliance should, hence, only be placed upon a careful 
microscopic examination. 

In all cases the fine, cheesy particles previously described should 
be carefully sought for, as they contain the largest number of bacilli. 
In their absence reliance should be placed upon the examination of a 
large number of preparations, attention being directed especially to 
the purulent and mucopurulent foci of the sputum. 

If but few bacilli are present the following procedure may be 
employed: About 100 c.c. of sputum are boiled with double the 
amount of water, to which from 6 to 8 drops of a 10 per cent, 
solution of sodium hydrate have been added, until a homogeneous 
solution has been obtained, water being added from time to time to 
allow for evaporation. The mixture is then centrifugated or set 
aside for twenty-four to forty-eight hours and examined for tubercle 
bacilli and elastic tissue. Or, the following procedure, suggested by 
d'Arrigo and Stampacchia, may be employed: Four or five sputum 
masses are placed in a test-tube and covered with Ranvier's acid 
alcohol (70 per cent, alcohol, containing 1 per cent, of concentrated 
hydrochloric acid), so that this fills about two-thirds of the tube. 
The mixture is Well shaken and kept, stoppered with cotton, for 
twenty-four hours at 37° C. or for three hours at 50° C. The acid 
alcohol destroys the mucus and fixes the cells and bacilli, which sink 
to the bottom. It is claimed that in a sediment prepared in this 
manner it is possible to demonstrate the tuberlce bacilli even after 
several years. 

If, notwithstanding the fact that all due precautions have been 
taken, no bacilli can be demonstrated in the sputum, and the clinical 
history and the physical signs are indefinite or negative, the proba- 
bilities are that we are dealing with a benign process. From an 
examination of the sputa alone in such cases it is utterly impossible 
to reach a definite conclusion. When the amount of sputum, more 
over, is small and contains but little pus, the absence of tuberlce 
bacilli in doubtful cases is less suggestive of the absence of tuber- 
culous disease than in cases in which the sputum is more abundant 
and mucopurulent. 

Only two bacilli are likely to be mistaken for the tubercle ba- 
cillus, viz., the bacillus of leprosy and the smegma bacillus. All 



BACTERIOLOGY OF THE SPUTUM 345 

three are characterized by the difficulty with which they take up 
basic dyes, and the great tenacity with which they hold the dye 
when once stained, even upon treatment w T ith mineral acids (acid 
fastness) and alcohol. This peculiarity has been generally referred 
to the presence of fat in the bacilli, but it appears from more recent 
researches that the chitin or chitinous substances in the bodies of the 
tubercle bacilli are primarily concerned in the reaction (Helbing). 1 
Sata 2 , moreover, has shown that other bacteria, such as the anthrax 
bacillus, the bacillus of glanders, the Staphylococcus aureus, etc., give 
a fat reaction which is as intense as that of the tubercle bacillus, 
while these organisms are not in the least resistant to the action of 
acids when stained. 

That confusion should arise in the differentiation between the 
tubercle bacillus and the bacillus of leprosy is very unlikely. More 
important is the smegma bacillus, which is known to occur at times 
upon the tonsils, the tongue, and in the tartar of the teeth of per- 
fectly healthy individuals. In sputum coming from the lungs it has 
been observed by Pappenheim, 3 Frankel, 4 and others. 

Methods of Staining the Tubercle Bacillus. 1. Gabbett's 
Method. — Bits of purulent or hemorrhagic material, or if present the 
cheesy particles referred to above, are spread on slides in thin layers. 
These are dried in the air and fixed by being passed a few times 
through the flame of a Bunsen burner or an alcohol lamp. The 
specimens are covered with a few drops of carbol-fuchsin solution 5 
and heated to boiling for one-quarter to one-half minute. The solu- 
tion is composed of 1 part of fuchsin dissolved in 100 parts of a 5 per 
cent, solution of carbolic acid and 10 parts of absolute alcohol. The 
excess of the staining fluid is drained off and replaced, without 
washing, with a solution, composed of 2 parts of methylene blue in 
100 parts of a 25 per cent, solution of sulphuric acid. After a 
minute or two they are washed in water, dried, and examined 
directly in oil. 

It has been suggested by Pagani 6 to use lactic acid instead of sul- 
phuric acid, in order to avoid a too energetic decolorization. He 
claims that excellent results are obtained if the second solution of 

1 " Erklarungsversuch f. d. specifische Farbbarkeit d. Tuberkelbacillen," 
Deutsch. med. Woch., 1900, V. B. p. 133. 

2 " Ueber d. Fettbildung durch verschiedene Bakterien," etc., Centralbl. f. allg. 
Path. u. path. Anat., 1900, Nos. 3, 4. 

" Befund v. Smegmabacillen im menschlichen Lungenauswurf," Berlin, klin. 
Woch., 1898, No. 37. 

4 " Einige Bemerkungen liber d. Vorkommen v. Smegmabacillen im Sputum," 
ibid., 1898, p. 880. 

5 In its place Czaplewsky recommends the use of a solution prepared by dis- 
solving 1 gram of fuchsin together with 5 c.c. of liquefied carbolic acid in 50 c.c. 
of glycerin and diluting to 100 c.c. with water. The solution does not give rise 
to the unsightly precipitates which are seen with the usual solution of carbol 
fuchsin, unless filtered. 

6 Ref. in Centralbl. f. Path. u. path. Anat., 1901, vol. xii, p. 323. 



346 THE SPUTUM 

Gabbet is replaced by the following: water, 50 c.c.; alcohol, 50 c.c. ; 
lactic acid, 2.5 grams; and methyl blue to saturation. The cover- 
glass specimens or slides are immersed in this solution for from 
fifteen to twenty seconds while gently agitating. 

Gabbet's method of staining is very convenient, and is the one 
most generally employed. The smegma bacillus, however, is also 
stained. 1 

2. The Weigert-Ehrlich Method. — Dried specimens are prepared, 
and stained for twenty-four hours with a solution of fuchsin in aniline - 
water. The staining fluid is prepared as follows: 

A test-tube full of water is shaken with about 20 drops of pure 
aniline oil and, after standing for a few minutes, filtered through 
a moistened filter. To this solution a few drops of a concentrated 
alcoholic solution of fuchsin or of methyl violet are added until the 
mixture becomes slightly cloudy — i. e., until a metallic lustre is noted 
on the surface. After twenty-four hours the preparations are washed 
with water in order to remove an excess of staining fluid. They are 
then immersed for several seconds in a dilute solution of nitric or 
hydrochloric acid (1 to 6, 1 to 3, or 1 to 2), and washed again with 
water or with absolute alcohol. At this time the specimens should 
have a faintly red or violet color. They are then dried, and 
mounted as usual. 

If it is desired to use a counter-stain, Bismarck brown, vesuvin, 
or methylene blue in watery solutions may be used. Into such a 
solution the specimen is placed after treatment with nitric acid and 
washing in water. It remains for about two minutes, and is then 
washed, dried, and mounted as above. 

3. Ziehl-Neelsen's Method. — A mixture of 90 parts of a 5 per 
cent, solution of carbolic acid and 10 parts of a concentrated alco- 
holic solution of fuchsin is used. The procedure is the same as that 
described under the Weigert-Ehrlich methd. It is usually not 
necessary to stain the preparations for twenty-four hours, however, 
and as a rule it is sufficient to place a few drops of the staining fluid 
upon the preparation and to heat over the free flame as described 
when the specimen is decolorized as before. In this manner excellent 
results may be obtained in a few minutes. 

Stained according to one of these methods, the bacilli appear as 
rods, measuring about 1.5 to 3.5 fi in length by 0.2 fi in breadth 
(Plate XVIII, Fig. 1). Much larger specimens may, however, also 
be seen, up to 11 fi in length. The shortest forms are commonly 
straight; the common types are usually slightly curved. They 
may occur joined in chains of two or three, and branching forms 
have also been observed. Occasionally one may see a couple of 
organisms, each bent to a crescent, linked in the form of the letter S. 

1 Frankel, Berlin, klin. Woch., 1884, vol. xxi, p. 195; and Deutsch. med. Woch., 
1887, vol. xvii, p. 552 



BACTERIOLOGY OF THE SPUTUM 347 

Very commonly they are beaded, and it is possible to make out 
from 1 to 8 clear spaces in an organism which are separated by 
round or rod-shaped granules, which are deeply stained and appear 
to lie in a lightly staining capsule. The small hyaline bodies were 
once regarded as spores, but it is more likely that they are vacuoles. 
Sometimes bacilli are seen which have club- or knob-shaped enlarge- 
ments at the extremities. These enlargements likewise have been 
viewed as spores, while others look upon them as products of degenera- 
tion. When present in large numbers, the bacilli are often seen in 
clumps, as though they had been agglutinated, but in every specimen 
isolated organisms are also found scattered through the field; or 
two or three in groups. 

Cultivation of the Tubercle Bacillus. —The cultivation of the 
tubercle bacillus is best accomplished on blood serum or glycerin agar 
(agar with 6 per cent, of glycerin added) at a temperature of 37° 
or 38° C. Below 30° C. and at a temperature higher than 42° C. 
the organism does not grow. Primary inoculation from the tissue 
should be made on blood serum, as the bacillus usually does not 
grow on glycerin agar when this is inoculated directly from the 
tuberculous focus. Subcultures, however, grow readily on glycerin 
agar and more rapidly than on blood serum. The individual colo- 
nies appear like small, dry scales, which gradually coalesce and form 
a wrinkled film of a dull, whitish color. Older cultures present a 
brownish or grayish-brown color. An adequate idea may be formed 
of the growth of the organism after two or three weeks. Sunlight 
rapidly kills the tubercle bacillus. 

Number in Sputum. — The number of bacilli which may be found 
in a sputum varies greatly, and while in general it may be said that it 
is in direct ratio to the intensity of the disease, and may thus be con- 
sidered of prognostic value, too much reliance should not be placed 
upon this statement, as in acute miliary tuberculosis, and in cases 
that have gone to the formation of cavities, the number may be small 
or they may be absent altogether. In an incipient case, on the other 
hand, in a little mucoid sputum the number may be large. If the 
number of bacilli steadily decreases in a series of examinations at 
intervals sufficiently long, the patient may be regarded as improv- 
ing, but here the constitutional symptoms and local signs give much 
more accurate information. 

If on repeated examination large numbers of tubercle bacilli are 
found, the disease has in all probability advanced to cavitation 
(Brown). 

In tabulating the number of tubercle bacilli in reports one may 
adapt Gaffky's scheme, modified by L. Brown as follows ( T V oil 
immersion; ocular 1; B. & L.): 

1. Only 1 to 4 in a whole preparation. 

2. Only 1 bacillus on an average in many fields. 



348 THE SPUTUM 

3. Only 1 bacillus on an average in each field. 

4. 2 to 3 bacilli on an average to each field. 

5. 4 to 6 bacilli on an average to each field. 

6. 7 to 12 bacilli on an average to each field. 

7. 13 to 25 bacilli on an average to each field. 

8. About 50 bacilli on an average to each field. 

9. 100 or more bacilli on an average to each field. 
10. Enormous numbers on an average to each field. 

An attempt has been made to attach prognostic significance to the 
form and grouping of the tubercle bacilli in the sputum. To judge 
from the experience gathered at Saranac, it appears that virulent 
and attenuated forms of tubercle bacilli possess practically the same 
morphology and that short bacilli usually represent a younger growth. 
Arrangement of the bacilli in clumps is more apt to be found in the 
severer cases, but may occur in all (Brown). 

Of the variations in number and form of the tubercle bacilli during 
treatment with Koch's tuberculin it is unnecessary to speak at this 
place, as the prognostic significance attaching to such variations is 
questionable. 1 

The Diplococcus Pneumoniae. — The Diplococcus pneumoniae of 
Frankel and Weichselbaum, also commonly termed the pneumococcus, 
is the recognized cause of acute croupous pneumonia in the majority 
of cases. It is then seen in the sputum in large numbers and recog- 
nized by its capsule. It may, however, also occur in the mouth of 
perfectly healthy individuals, so that its diagnostic significance is 
somewhat limited. To demonstrate the organism smears on slides 
or cover-glasses are placed for one or two minutes in a 1 per cent, 
solution of acetic acid ; they are then removed and the excess of acetic 
acid drawn off, when they are allowed to dry in the air; they are 
subsequently placed for several seconds in saturated aniline-water 
and gentian-violet solution, washed in water, and examined. Rod- 
shaped diplococci (Fig. 123), surrounded by a capsule, which latter 
is considered the characteristic feature of this organism, will be seen 
in cases of acute croupous pneumonia. 2 

As a rule the capsule is not well shown in this way. The best 
results are obtained with Buerger's method? Smears are prepared 
as usual. As soon as the edges begin to dry they are covered with 
Miiller's fluid, 4 saturated with bichloride of mercury (ordinarily about 
5 per cent.). The specimens are gently warmed over the flame for 

1 F. Fischel, Unters. iiber d. Morphol. u Biol. d. Tuberculose-Erregers, 1895. 
Gaffky, Mittl. aus. d. Kais. Gesundh. Anz., vol. xi, p. 126; L. Brown, Jour. 
Amer. Med. Assoc, 1903, vol. xl, p. 514. 

2 Frankel, Zeit. f. klin. Med. 1886, vol. ii, p. 437. Weichselbaum, Wien. med. 
Woch., 1886, vol. xxxix, pp. 1301, 1339, 1367. 

3 Buerger, L. Med. News., Dec. 10, 1904. 

4 Composition of Miiller's fluid: 2.5 grams potassium bichromate, 1 gram 
sodium sulphate, and 100 c.c. of water. 



BACTERIOLOGY OF THE SPUTUM 349 

about three seconds (using cover-glass smears), rapidly washed in 
water, flushed once with alcohol (SO to 95 per cent.), and then treated 
with ordinary tincture of iodine for one or two minutes. The iodine 
in turn is thoroughly washed off with alcohol and the preparations 
dried in the air. They are then stained for two to five seconds with 
gentian aniline-water (aniline-oil 10 c.c, water 100 c.c; shake, filter, 
and add 5 c.c. of a saturated alcoholic solution of gentian violet; or 
10 per cent, aqueous fuchsin solution, viz., saturated alcoholic solution 
of fuchsin 10 c.c. and water 100 c.c). Washing with a 2 per cent, 
aqueous solution of salt completes the process. The preparations 
are examined in a drop of the salt solution and ringed with vaselin. 
With this method there is visible a refractile, deeply staining, 
regularly outlined, narrow, elliptical capsule membrane, separated 
from the diplococcus by a clear area of capsular substance which 
either remains unstained or takes a faint color. 



Fig. 123. — Pneumococcus from bouillon culture, resembling streptococcus. (Park.) 

If smears are to be made from cultures or from material which in 
itself is essentially non-albuminous, Buerger directs that a drop of 
blood serum diluted with an equal amount of saline solution should 
be placed upon the slide or cover, and that the smear be made in this. 
Epstein finds that albumen-water (egg albumen shaken with an equal 
volume of water or normal salt solution) works just as well and 
will keep for two or three weeks. 

The Bacillus of Influenza. — The bacillus of influenza was discovered 
in 1892 by Pfeiffer. It is found in the bronchial sputum in large 
numbers and is essentially characterized by its minute size, measuring 
only 0.2 to 0.3 fi in breadth by 0.5 fi in length (Fig. 124). The organ- 
isms occur for the most part singly, but may also form chains of 
threes and fours. In suitably stained specimens they may at first 
sight appear as diplococci, owing to the fact that the poles are stained 



350 THE SPUTUM 

more deeply than the intervening portion. Carbol fuchsin diluted 
in the proportion of 1 to 10 with water stains the bacillus very well 
and brings out the polar staining. 

The organism is non-motile and forms no spores. It can be grown 
on media containing blood or serum (blood agar, hydrocele agar, 
Loffler's serum). Human blood and pigeon blood are the best. 
Growth, however, in any event is slight and occurs slowly. In order 
to cultivate the influenza bacillus from the sputum, this is collected 
in sterile cups and examined without delay. The sputa are washed 
in sterile bouillon or sterile normal salt solution and cultures made on 
blood agar. (Boggs 1 recommends pigeon-blood agar or agar to which 
sterile fetal blood has been added.) Tiny, water-clear colonies then 
develop, as described by Pfeiffer. On the fetal-blood agar Boggs 
noted that involution forms appear earlier and in much greater num- 
ber than when pigeon, rabbit, or adult 
human blood was used. Some of these 
forms are so large and irregular as to give 
at first sight the impression of a mixed 
infection. 

From the blood the organism is rarely 
obtained. 

Influenza-like bacilli have been found in 

whooping-cough sputa by Spengler, Joch- 

mann, and Krause, and more recently by 

fig. i24.-^nTm^za bacilli. Wollstein. The organism in question has 

been named the Bacillus pertussis, Eppen- 

dorf. According to Spengler the bacillus of Czaplewski and Hensel 

is only a contaminating pseudodiphtheria bacillus. 

To cultivate the Bacillus pertussis the sputum masses coughed up 
after a paroxysm are washed in six successive beakers of peptone 
water and spread upon blood-agar plates prepared by mixing placental 
blood with melted agar. The predominating colonies are then small, 
transparent, dew-drop like, and not surrounded by a hemolytic zone, 
as in the case of the pneumococcus and streptococcus. Microscopic- 
ally they appear as slightly raised, almost structureless droplets. 
After forty-eight hours the colonies show a slightly granular centre. 
The bacilli also grow in bouillon to which a drop of fresh or hemolyzed 
blood is added. On ascitic fluid agar, glycerin agar, Loffler's serum, 
plain bouillon, serum broth, milk and gelatin no growth takes place. 
The organisms are not motile. They are short, plump, ovoid, 
with rounded ends, lying singly or in small groups between the pus 
and epithelial cells of the sputum They are decolorized by Gram's 
method. Somewhat larger forms are found in older cultures, and 
Spengler speaks of very long chains. 

1 Amer. Jour., Nov., 1905, p. 902. 




BACTERIOLOGY OF THE SPUTUM 35I 

Wollstein 1 obtained agglutination with the serum of the correspond- 
ing child in dilutions of 1 to 200 and occasionally of 1 to 500. 

The Smegma Bacillus. — In a few isolated cases the smegma bacil- 
lus has been encountered in the sputum, and, as I have already 
stated, the same organism may normally be present in the saliva, 
the coating of the tongue, the tartar of the teeth, etc. Like the 
tubercle bacillus, it resists the decolorizing action of acids when 
once stained, and may hence be confounded with it unless special 
precautions are observed (see Urine). 

The Typhoid Bacillus. — It has been conclusively shown that the 
typhoid bacillus can be present in the sputum of typhoid patients, 
especially if there is a coexistent bronchitis or pneumonia. 2 

The Plague Bacillus. — The plague bacillus is seen in the sputum 
in enormous numbers in cases of the pneumonic type of the disease. 
By direct observation, however, it may not be recognized immediately, 
and it is best in every case to resort to culture as well (Fig. 44, page 
176, see Blood). The organism ma\ be found in the sputum on the 
first day of the disease. 

Micrococcus Catarrhalis. — This organism is frequently seen in the 
sputa and nasal discharge. It is larger than the common staphylo- 
cocci, but, like these, frequently occurs in lateral pairs, the contiguous 
sides being concave. 

Micrococcus Tetragenus. — This organism is frequently seen in the 
sputum under the most varied pathological conditions and may also 
occur in the mouths of perfectly healthy individuals. It is a coccus 
occurring in fours, each measuring about 1 // in diameter. The form 
which is found under normal conditions, in contradistinction to dis- 
ease, cannot be cultivated. 

Staphylococci and Streptococci may be found in the mouths of 
apparently healthy individuals, but are more commonly encountered 
in inflammatory conditions of the most divers kinds. Where cavity 
formation is going on in the lungs they are usually very numerous. 

Streptothrices. — Within recent years there is a tendency among 
pathologists to abandon the older terms actinomyces, cladothrix, etc., 
and to speak of infections with branching mycelial organisms under 
the collective term streptothricosis, designating the specific variety by 
its special term. 

Up to 1902 about 100 cases of supposed cattle actinomycosis had 
been reported in the United States, as occurring in man (Ewing), 
but it is difficult to say how many of the older cases really belonged 
to this order; in the light of recent investigations it seems not 
unlikely that many were referable to different species. 

In the cattle disease yellow granules (so-called sulphur granules) 
may be found in the pus derived from actinomycotic tumors, in the 

1 Jour. Exper. Med., 1905, vol. vii, p. 335. 

2 M. W. Richardsen, Boston Med. and Surg. Jour., Feb. 5, 1903. 



352 



THE SPUTUM 



sputum, and in the feces, when the disease has attacked the lungs and 
intestines respectively, which measure from 0.5 to 2 mm. in diameter. 
If such a granule is examined microscopically , slight pressure being 
applied to the cover-glass, it will be seen to consist of numerous threads 
which radiate from a centre in a fan-like manner and present club- 
shaped extremities (Fig. 125). 

The cattle organism is termed the Streptothrix (Actinomyces) bovis 
communis (Streptothrix aetinomycotica, or ray fungus). It may be 
demonstrated in the following manner: Dried cover-glass preparations 
are stained for five to ten minutes with aniline- water — gentian violet 
(see Weigert-Ehrlich stain for tubercle bacilli), when they are rinsed 
in normal salt solution, dried between filter paper, and transferred 
for two or three minutes to a solution of iodopotassic iodide (1 to 100 





Fig. 125. — Actinomyces. (Musser 



or 1 to 150). They are then again dried between layers of filter paper, 
decolorized in xylol-aniline oil (1 to 2), washed in xylol, and mounted in 
balsam. The mycelium assumes a dark-blue color. 1 The organism 
is acid fast, but loses its color on washing with alcohol (95 per cent.). 

In addition to the cattle cases there exists a group of pulmonary 
cases which present the clinical features of tuberculosis, broncho- 
pneumonia, or gangrene, but in which the infecting agent is a species 
of streptothrix different from the cattle variety. About 30 cases of 
this kind have been reported (1906). Different species have been 
described, such as the Streptothrix eppingeri (Cladothrix asteroida), 
Streptothrix pseudotuberculosa, Flexner; Streptothrix hominis, Fouler- 
ton, and Streptothrix isra^li. 

The organism is found in the sputum, often in the form of small, 
grayish-yellow granules. These are made up of a mycelium of 
branching organisms, which in the unstained specimen appear as fine, 
homogeneous, glistening threads, about two to four times as wide as a 
tubercle bacillus. They are acid fast, but can be decolorized with 



R. Paltauf, Sitzungsber. d. K. K. Gesellsch. d. Aerzte Wien, 1886. 



BACTERIOLOGY OF THE SPUTUM 



353 



alcohol. In such specimens many of the threads present a beaded 
appearance and sometimes seem to be breaking up into short rods of 
varying length. With Gram some varieties stain well, while others 
do less so. Culture yields uncertain results. Flexner obtained no 
growth. Eppinger succeeded with gelatin, inspissated horse serum, 
maltose agar, and potato. 

Literature. — Ashton and Norris, Jour. Amer. Med. Assoc. Sept. 9, 1905. 
Flexner, Trans. Assoc. Amer. Phys., 1898, vol. xiii. Warthin and Olney, Amer. 
Jour., Oct., 1904. W. G. Ewing, Johns Hopkins Hosp. Bull., 1902, vol. xiii. J. 
Ruhrah, Annals of Surg., 1899, vol. xxx (analysis of 62 cases). 

Blastomycetes. — In the rare cases of systemic blastomycosis blasto- 
mycetes may be demonstrable in the sputum. Such a case has 



, ,, m* " m 


= 


^•ofcfV/"" 






o * 


"\'„ *•* j&^ik j* 





Fig. 126 —Blastomycetes. Smear from sputum mounted in 1 per cent, potassium hydrate 
solution, showing circular and budding organisms. X 1200. (Eisendrath and Ormsby.) 

been described by Eisendrath and Ormsby. 1 For the examination of 
pus or sputum the writers recommend the addition of a little 10 per 
cent. NaOH solution to the specimen and to examine unstained with 
a|orj objective. The refractile parasite is thus well brought out. 
(Figs. 126, 127, and 128.) 

Molds. — Of other fungi which are occasionally observed, there 
may be mentioned various varieties of mucor and aspergillus. Some 
of these organisms (Mucor corymbifer and Aspergillus fumigatus) 
have been found associated with cavity formation and seem to have 



23 



1 Journal Medical Association, October 7, 1905. 



354 



THE SPUTUM 



pathogenic properties. They may at times overgrow the saprophytic 
bacilli (Pneumonomycosis aspergillina, sell mucorina). They are best 
studied in the fresh specimen, not stained (Figs. 129 and 130). 

Sarcina pulmonalis has been found at times, especially in the 
mycotic bronchial plugs occurring in putrid bronchitis. It is usu- 
ally smaller than the Sarcina ventriculi, but larger than the variety 
observed in the urine; it presents the characteristic form of the latter. 



3M 



mm 





'-* J 






.-'"'■' / : 










"' a' 


t; *. *' 










1 V^ i',— 






* 


^t* 


^& 


'Z-^'*- 








t • ^ ; 


~'-.'\. ■ ' 






^^>w*. 


• Sja&iS^V"^ 


~'~~ : '>2^ ' 






"-•-_' ^v**-^ 


J££*.""& - 


■ \\~ ' : "~ ~ 


".. ~- N *>v 




- \ 



Fig. 127. — Blastomycetes. Smear from growth on media, five weeks old, in 1 per cent, potassium 
hydrate solution. Low power. (Eisendrath and Ormsby.) 

Oidium albicans may be seen in children, and is usually derived 
from the mouth. 

Crystals. — Of crystals which may occur in sputa, it will be neces- 
sary to consider briefly the crystals of Charcot-Leyden, hematoidin, 
cholesterin, margarin, tyrosin, calcium oxalate, and triple phosphates. 

Charcot-Leyden Crystals. 1 — These crystals were discovered in the 
sputa of patients suffering from bronchial asthma, and were supposed 

1 Leyden, Virchow's Archiv, 1872, vol. liy, p. 324. Schreiner, Liebig's Annal., 
1878, vol. cxciv, p. 68. Cohn, Centralbl. f. allg. Path. u. path. Anat., vol. x, 
p. 940. Brown, Phila. Med. Jour., 1898, p. 1076. 



BACTERIOLOGY OF THE SPUTUM 



355 



to stand in a causative relation to the disease. This view has been 
abandoned, and it is known that they may occur in other diseases as 
well. But while their presence is almost constant in bronchial asthma 
at a time when Curschmann's spirals can also be demonstrated, they 
are only exceptionally met with in other diseases, such a;s acute and 
chronic bronchitis, phthisis, etc. They were formerly regarded as 
identical with Bottcher's sperma crystals, but it has been shown that 
this is not the case. They are straight, hexagonal, double pyramids, 




r, p) Kk^ , 



W 

f SfA 



Fig. 128. — Higher magnification of Fig. 127. X 1200. 

and appear under the microscope as flattened needles of variable size 
(Fig. 112). Some attain a length of from 40 // to 60 fi, while others are 
scarcely visible even with a comparatively high power of the micro- 
scope. They show a feeble, positive, double refraction, and have but 
one optical axis, while the sperma crystals are biaxial and strongly 
double refracting. Their behavior to solvents is essentially the 
same as that of the sperma crystals, but they differ from these in 
their insolubility in formol. They are colored yellow with Florence's 



356 



THE SPUTUM 



reagent, while the sperma crystals are stained a bluish black. Very 
curiously the appearance of Charcot-Leyden crystals is closely asso- 
ciated with the presence of eosinophilic leukocytes, and they have 
hence been termed leukocytic crystals. They may in fact originate 
within the cells. In bronchial asthma it is not uncommon to find 
microscopic preparations of the sputum literally studded with 
eosinophilic leukocytes and free granules. Outside the sputum they 
are also found in the blood, in myelogenous leukemia, and in the stools 
in association with animal parasites. They readily form in both 
normal and abnormal red bone-marrow, and excellent specimens 
may be obtained for purposes of demonstration if a piece of a rib is 
allowed to remain exposed to the air for a few days. The marrow 
then usually contains large numbers. The crystals also form in 
decomposing viscera in general, and at times form a complete covering 

of old anatomical preparations. 
Their occurrence may be re- 
garded as evidence of retrogres- 
sive changes in the cellular 
elements of an organ. Of the 
relation which they bear to the 
eosinophilic leukocytes, with 
which they are so constantly 
associated, nothing is known. 
The Charcot-Leyden crystals 
can be stained with the triacid 
stain, with thionin, with the 
eosinate of methylene blue, and 
other dyes. 

Hematoidin crystals may be 
observed in the sputa following 
extravasations of blood into the 
lung. They frequently occur in the form of ruby-red columns or 
needles; amorphous granules, however, are also seen, enclosed in the 
bodies of leukocytes, in which case they are probably always indica- 
tive of a previous hemorrhage, while the needles are generally 
observed when an abscess or empyema has perforated into the lungs. 
The substance is derived from blood pigment, and is now known 
to be identical with bilirubin. 

Cholesterin crystals are at times seen in the sputa in cases of 
phthisis, pulmonary abscess, and, in general, whenever old accumula- 
tions of pus have entered the lung from a neighboring organ. They 
are reaclily recognized by their characteristic form and chemical 
properties (see Feces). 

Fatty acid crystals are frequently observed in cases of putrid bron- 
chitis and gangrene of the lung, and also in cases of bronchiectasis 
and phthisis. They occur in the form of single needles or groups 




Fig. 129.- 



- Aspergillus fumigatus 
(Frankels.) 



350. 



BACTERIOLOGY OF THE SPUTUM 



357 



of needles, which are long and pointed. They are easily soluble in 
ether and hot alcohol; insoluble in water and acids. Chemically, 
they are probably composed of the higher fatty acids, such as palmitic 
and stearic acids. 

Tyrosin crystals have been observed in cases of putrid bronchitis, 
perforating empyema, etc. Leucin is then usually also present, occur- 
ring in the form of highly refractive globules. For the recognition 
of these bodies, particularly of tyrosin, a chemical examination should 
always be made, as crystals of the soaps of fatty acids have frequently 
been mistaken for those of tyrosin (see Urine). 

Calcium oxalate crystals are rarely seen. Fiirbringer observed 
them in large numbers in a case of diabetes, and Unger found them 
in a case of asthma. They are readily recognized by their envelope 




Fig. 130. — Aspergillus fumigatus of the lung, partly schematic: a, mycelium of aspergillus 
in roset-like rays; b, sporangium. X 285. (Weichselbaum.) 



form and central cross, but they occur also in amorphous masses. 
They are soluble in mineral acids; insoluble in water, alkalies, organic 
acids, alcohol, and ether. 

Triple phosphate crystals also are rarely seen, but may occur in 
cases of perforating abscesses, etc. They are recognized by their 
coffin-lid shape and the readiness with which they dissolve in acetic 
acid. 

The Pneumoconioses. Anthracosis. — To some extent particles of 
carbon may be found in the sputum of almost every individual. The 
expectoration in such cases is of a pearl-gray color, and is brought 
up in larger or smaller masses, especially in the morning upon rising. 
Larger amounts are noted in miners and in those who are brought 
into close contact with coal-dust. Microscopically, particles of car- 



358 THE SPUTUM 

bon and epithelial cells, of the alveolar type, as well as leukocytes 
loaded with the pigment, are seen. 

Siderosis. — In siderosis the sputum presents a brownish-black 
color and contains cells enclosing particles of ferric oxide. These 
may be readily recognized by treating with a drop of ammonium 
sulphide or potassium ferrocyanide solution in the presence of hydro- 
chloric acid, when a black color on the one hand or a blue color on 
the other is obtained in the presence of iron. 

Chalicosis. — In chalicosis silicates are found in the sputa. 1 

CHEMISTRY OF THE SPUTUM. 

In addition to the substances described, sputum contains certain 
albumins, volatile fatty acids, glycogen, ferments, and various inor- 
ganic salts. 

Among the albumins may be mentioned serum albumin, and 
especially mucin, which is often present in large amounts. In pneu- 
monic and purulent sputa albumoses also have been found. 

In order to demonstrate the presence of serum albumin the sputa 
are treated with dilute acetic acid, when the filtrate is tested with 
potassium ferrocyanide, as described in the chapter on Urine. Serum 
albumin is, of course, found in notable quantities in cases of edema 
of the lungs. Especially interesting is the albuminous expectoration 
which at times follows thoracentesis. The amount of sputum usually 
varies between 200 and 900 grams, but may be much larger and may 
reach 2000 c.c. or even more. Occasionally it begins before the tap- 
ping is completed or immediately after. More commonly, however, 
an interval varying from five minutes to one or two hours elapses 
before the expectoration begins. Its duration is variable. Sometimes 
it lasts only a few minutes, more often an hour or two, and in rarer 
cases a whole day or two. The condition is probably due to edema 
of the lungs. 2 

The volatile fatty acids contained in sputa may be obtained by 
diluting with water, acidifying with phosphoric acid, and distilling, 
when the distillate is further examined as described in the chapter 
on Feces. Acetic, butyric, propionic, and capronic acids have been 
found. 

The fats and fixed fatty acids are extracted from the residue with 
ether, and shaken with a solution of sodium carbonate in order to 
transform them into their sodium salts, when the ether is decanted 
and evaporated, leaving the soaps behind. 

1 Betts, ''Chalicosis Pulmonum," Jour. Amer. Med. Assoc, 1900, No. 2. 

2 In the United States cases of albuminous expectoration following thoracentesis 
have peen reported by Pepper, Allen, Pateck, and Riesman. See especially the 
paper by Riesman, in which a full account of the literature is given. Amer. 
Jour. Med. Sci., April, 1902, p. 620. 



CHEMISTRY OF THE SPUTUM 359 

Glycogen has repeatedly been demonstrated in sputa, and may be 
detected by Ehrlich's method (see Blood). 

The sputa of gangrene of the lung and putrid bronchitis have been 
shown to contain a ferment resembling trypsin. In order to test for 
this, the sputa are extracted with glycerin; the examination is then 
continued as described in the chapter on the Examination of Cystic 
Contents. 

The myelin granules, as I have already indicated, consist largely 
of protagon, lecithin, and cholesterin. 



CHAPTER VII. 

THE URINE. 

GENERAL CHARACTERISTICS OF THE URINE. 

Appearance. — Normal urine, just voided at an ordinary tempera- 
ture, is either perfectly clear or but faintly cloudy, owing to the fact 
that the acid and normal salts present are all soluble in water. It may 
be stated, as a general rule, that whenever a urine freshly passed 
presents a distinct cloudiness, some abnormality exists. 

When allowed to stand for a time a light cloud develops, which 
gradually settles to the bottom, constituting the so-called nubecula 
of the ancients. Examined under the microscope this is found to 
contain a few round, granular cells, somewhat larger than normal 
leukocytes, the so-called mucous corpuscles, and a few pavement- 
epithelial cells, derived from the bladder or genital organs. Chemi- 
cally the nubecula probably consists of traces of mucus. 

When kept for twenty-four hours at an ordinary temperature, 
crystals of uric acid are frequently observed in addition to the above 
elements, usually presenting the so-called whetstone form. If, how- 
ever, the temperature at which the urine is kept approaches the freezing 
point, the entire volume becomes cloudy, owing to precipitation of 
acid urates, as these are much less soluble in cold than in warm water; 
on standing they gradually settle to the bottom of the vessel and form 
what -is known as a sediment, while the supernatant fluid again becomes 
clear. 

If kept still longer exposed to the air, at the temperature of the 
room, the entire volume of urine again becomes cloudy, owing to a 
diminution of its normal acidity, the result being a precipitation of 
ammonio-magnesium phosphate, calcium phosphate, and still later, 
when the urine has become alkaline, of ammonium urate. 

Gradually a heavy sediment, containing these salts in addition to 
the constituents of the primitive nubecula, forms at the bottom of the 
vessel; the supernatant fluid, however, remains cloudy. On micro- 
scopic examination it will be seen that this cloudiness is due to the 
presence of enormous numbers of bacteria. 

The changes which take place in a normal urine when allowed to 
stand at ordinary temperature may be tabulated as follows: 



GENERAL CHARACTERISTICS OF THE URINE 361 

1. Urine clear, no sediment; reaction acid. 

2. Urine slightly cloudy, owing to development of the nubecula; 

reaction acid. » 

tvt -, , f Mucous corpuscles, 
Nubecula j Pave ment-epithelial cells. 

3. Urine clear; the nubecula has settled; reaction acid. 

f Mucous corpuscles, 

Sediment \ uricScryst'als, 
[_ A few bacteria. 

4. Urine cloudly, owing to the precipitation of phosphates; reaction 

faintly acid or alkaline. 

5. Urine cloudy, owing to the presence of bacteria; reaction alkaline. 



Bacteria, 

Mucous corpuscles, 



Sediment \ S»al eeHs 



Triple phosphates, 
Tricalcium phosphate, 



t Ammonium urate. 

Color. — The color of normal urine may vary from a very light yel- 
low to a brownish red, the particular shade depending essentially upon 
the specific gravity, becoming lighter with a diminishing and darker 
with an increasing density. Pathologically the same rule holds good, 
except in diabetes, in which a very high specific gravity is generally 
associated with a very light color. The reaction of the urine also 
exerts a marked influence upon its color, an acid urine being more 
highly colored than an alkaline urine, which can be readily demon- 
strated by allowing a specimen of acid urine to become alkaline, and 
by treating an alkaline urine with dilute hydrochloric or acetic acid. 
At the same time it may be said that every urine darkens slightly on 
standing, the reaction remaining acid. 

The various shades observed in normal urines may be grouped 
under the following headings: 

1. Pale urines vary from a faint yellow to a straw color. 

2. Normally colored urines are of a golden or an amber yellow. 

3. Highly colored urines present a reddish-yellow to a red color. 

4. Dark urines vary between brownish red and reddish brown. 
As these shades may occur in both normal and pathological urines, 

definite conclusions cannot, as a rule, be drawn from mere inspection. 
A very pale urine indicates an excess of water, which may be 
normal, but may also occur in such diseases as chronic interstitial 
nephritis, diabetes mellitus, diabetes insipidus, hysteria, and the 
various anemias; it is further seen during convalescence from acute 
febrile diseases, while a highly colored urine, though also occurring 
in health, may indicate the existence of a febrile process. 

The normal color of the urine is probably owing to the presence 



362 THE URINE 

of several pigments, which are most likely closely related to each 
other and to hematin. 

In addition to these colors others may be observed at times which 
are either pathological or accidental — i. e., due to the presence of cer- 
tain drugs. The former are, on the whole, of greater importance to 
the physician than those mentioned above, as more definite conclu- 
sions can be drawn from their presence. The most important 
pathological pigments are: 

1. Blood-coloring matter. The color in such cases may vary from 
a bright carmine to a jet black, the exact shade depending upon the 
quantity of blood-coloring matter present, upon changes that the 
blood may have undergone either before or after being passed, and 
also upon the presence of the pigment in solution or contained in red 
corpuscles. 

2. Biliary coloring matter. The color here varies from a greenish 
yellow to a greenish brown. 

Among the accidental abnormalities in color are those due to the 
presence of substances like carbolic acid and its congeners, santonin, 
etc. A milky-colored urine is observed in cases of chyluria. 

As the recognition of the causes of such alterations, normal, 
pathological, and accidental, largely depends upon a more detailed 
study of the individual pigments, this subject will be dealt with 
more fully farther on. (See Pigments and Chromogens.) 

Odor, — The odor of the urine is usually of little significance. 
Normally it resembles that of bouillon, and in some cases that of 
oysters; it is probably due to the presence of several volatile acids. 
The odor of urines undergoing decomposition is characteristic and 
has been termed "the urinous odor of urine, " an ill-chosen term, as 
this odor is always indicative of an abnormal condition. 

The ingestion of asparagus, onions, oil of turpentine, etc., pro- 
duces characteristic odors. 

Consistence. — Urine, while normally fluid and but slightly viscid, 
may in disease acquire a marked degree of viscidity, which becomes 
especially apparent upon attempting its filtration; the liquid passes 
through the paper with more and more difficulty, and finally clogs its 
pores altogether. In old, neglected cases of cystitis it may be ropy 
and gelatinous. 

Quantity, — The quantity of the urine is normally subject to great 
variations, the amount eliminated in the twenty-four hours being 
influenced by that of the fluid ingested, the nature and quantity of the 
food, the process of digestion, the blood pressure, the surrounding 
temperature, sleep, exercise, body weight, sex, age, etc. 

It is easy to understand, then, why figures given by different 
observers in different countries should vary considerably. Salkow- 
ski, in Germany, thus gives 1500 to 1700 c.c. as the normal amount; 
v. Jaksch, in Austria, 1500 to 2000 c.c; Landois and Sterling, in 



GENERAL CHARACTERISTICS OF THE URINE 363 

England, 1000 to 1500 c.c; Gautier, in France, 1250 to 1300 c.c. 
In the United States I have found an average secretion of from 1000 
to 1200 c.c. in the adult male, and 900 to 1000 c.c. in the adult female. 
It is thus seen that the secretion of urine is greatest in Germany and 
Austria, where the body weight and ingestion of liquids are greater 
than in England, France, and the United States. 

Children pass less, but relatively more (considering their body 
weight) urine than adults. 

Women pass somewhat less than men. 

During the summer months, when a larger proportion of water 
is eliminated through the skin and lungs than in cold weather, less 
urine is voided. The same occurs during repose, more urine being 
passed during active exercise, and hence less during the night than 
during the day. 

The amount of urine secreted in the different hours of the day 
varies greatly, reaching its maximum a few hours after meals. It 
decreases toward night, and reaches its lowest point in the first hours 
of the night, after which it begins to rise rapidly until 2 or 3 o'clock 
in the morning. 

The ingestion of large amounts of liquid, of course, increases the 
daily amount considerably, and 3000 c.c. may be passed under such 
conditions by an individual in good health, while it may decrease to 
800 or 900 c.c. when but little liquid is taken. 

After the ingestion of much solid food the secretion of urine is 
temporarily diminished. 

Water containing no salts possesses distinct diuretic properties, as 
do also beer, wine, coffee, tea, etc. 

The most important medicinal diuretics are digitalis, squill, broom, 
spirit of nitrous ether, juniper, urea, etc. 

Pathologically the amount of urine varies within wide limits. In 
a given case, moreover, it may be exceedingly difficult to determine 
whether or not the secretion is within physiological limits. As a 
general rule, whenever less than 500 c.c. or more than 3000 c.c. are 
passed some abnormal condition exists, providing all other causes 
which might lead to the secretion of such an amount can be elimi- 
nated. 

Clinically we speak of polyuria and oliguria. 

Polyuria. — Polyuria is observed in many diseases, and is present 
under such varied conditions that a classification is only warrantable 
upon a hypothetical basis, especially as the causative factors concerned 
in its production are mostly unknown. 

As polyuria is almost invariably associated with diabetes mel- 
litus, its presence in any case should always excite suspicion and 
lead to a proper examination. The quantity of fluid eliminated in 
diabetes is usually dependent upon the amount ingested. The excre- 
tion of a proportionately large amount of fluid, however, does not 



364 THE URINE 

necessarily follow the ingestion directly, and retention of a large 
amount may occur; it has been shown, as a matter of fact, that the 
diabetic patient excretes liquids with greater difficulty than the 
healthy subject. At the same time it should be borne in mind that 
the polyuria in diabetes is not necessarily continuous, and that 
periods during which a normal or even a subnormal amount is 
observed may alternate with true polyuria. From 2 to 26 or even 
50 liters may be passed within twenty-four hours. Intercurrent dis- 
eases of a febrile character may modify the quantity very materially 
and cause the elimination of a normal or subnormal amount. The 
cause of the polyuria in diabetes mellitus is unknown. 

The polyuria associated with the resorption of large pericardial, 
pleural, ascitic, and subcutaneous effusions is more readily under- 
stood, although the primum mobile may be unknown; it depends 
in such cases entirely upon the presence of excessive quantities of 
fluid in the bloodvessels. 

A form of polyuria which has been termed "epicritic polyuria" 
is frequently observed during convalescence from acute febrile dis- 
eases, and is of prognostic importance. Its occurrence in a given 
case is regarded by many as a good omen, especially in typhoid 
fever; still it must not be forgotten that a polyuria may occur after 
subsidence of the fever, and be followed by a considerable degree of 
oliguria, and in some cases may precede death. A polyuria of this 
kind probably always indicates the elimination of waste products 
which have accumulated in the blood during the course of the disease, 
but it may, at the same time, be due to the presence of retained water. 

Second in constancy is the polyuria associated with granular 
atrophy of the kidneys. Cases have been reported in which 10,000 
c.c. of urine were secreted in the twenty-four hours; 2000 to 4000 
c.c. represent the usual amount. 

Polyuria is of frequent occurrence early in the course of renal tuber- 
culosis, the increase amounting to one-half of the normal amount. 

Very curiously, polyuria may occur also in association with mul- 
tiple myelomas of the bones and the presence of Bence Jones' 
albumin in the urine. In one of the cases reported by Hamburger/ 
which I had occasion to study in greater detail from a chemical 
point of view, 3500 c.c. were voided in the twenty-four hours. The 
symptom, however, is not constant. 

Polyuria, furthermore, has been observed in the most diverse 
diseases of the nervous system, both functional and organic. It is 
frequently observed both as a transitory and a more or less per- 
manent symptom in cases of hysteria. Large quantities of a very 
pale urine are secreted after the occurrence of severe hysterical 

1 "Two Examples of Bence Jones' Albuminuria associated with Multiple Mye- 
loma," Johns Hopkins Hosp. Bull., Feb., 1901. C. E. Simon, Amer. Jour. Med. 
Sci., 1902, vol. cxxiii, p. 954. 



GENERAL CHARACTERISTICS OF THE URINE 365 

seizures, but the same may be observed throughout the course of 
the disease. A similar condition is frequently seen in neurasthenia, 
migraine, chorea, and epilepsy. 

Generally speaking, it may be said that a paroxysmal polyuria in 
nervous diseases is associated with functional derangement, while 
a continuous polyuria appears to be connected rather with true 
organic changes. It has been observed in certain cases of tabes, 
cerebrospinal and spinal meningitis; during the first stage of general 
paresis; in association with tumors involving the medulla, the cere- 
bellum, and the spinal cord; in injuries affecting the central nervous 
system, in Basedow's disease, etc. Cases of idiopathic diabetes 
insipidus also should probably be classified under this heading. 
Enormous quantities of urine may be secreted in this disease, which 
are equalled only by cases of diabetes mellitus, and may at times 
reach 43 liters per diem. 

Oliguria. — Oliguria is, on the whole, more frequent than polyuria, 
and is met with in almost all conditions associated with a lowered 
blood pressure. First in order stand those cases of cardiac disease 
in which compensation has failed, whether the cardiac weakness is 
primary or occurs secondarily to other diseases — i. e., pulmonary, 
hepatic, and renal. 

The oliguria observed in the so-called continued fevers, notably 
typhoid fever, is probably also referable to cardiac weakness. It 
should be remembered, however, that a larger proportion of water 
is eliminated through the skin and lungs than normally, and that 
a retention of fluids also undoubtedly occurs which is not due to 
cardiac weakness; still other factors may be concerned in its pro- 
duction. 

The oliguria occurring in acute nephritis and in chronic paren- 
chymatous nephritis in all probability depends largely upon mechani- 
cal causes, the increased intra-canalicular resistance in the form of 
desquamated epithelium and tube casts, as well as the pressure of 
the exudate upon the bloodvessels obstructing the passage of urine, 
while the functional activity of the diseased glandular elements is at 
the same time lowered. Upon mechanical causes, also, depend all 
those cases of oliguria which are associated with the presence of a 
stone or tumor pressing upon a portion of the urinary tract. 

Oliguria may occur as a nervous manifestation in connection with 
puerperal eclampsia, lead colic, hysteria, psychic depression, preced- 
ing and during epileptic seizures, etc. Whenever there is a diminu- 
tion in the amount of bodily fluids oliguria is also observed; this is 
particularly marked in cholera and following severe hemorrhage. 

Obstruction to the flow of blood in the vena cava or liver, lead- 
ing to an increase of venous pressure and a decrease of arterial 
pressure in the kidneys, likewise results in oliguria, as is seen in 
atrophic hepatic cirrhosis, acute yellow atrophy, thrombosis of the 



366 THE URINE 

vena cava and the renal vein, or in cases in which pressure is exerted 
upon these by tumors, ascitic fluid, etc. 

In any case the oliguria may go on to complete anuria, which 
condition not infrequently precedes death. Anuria may, however, 
also occur independently of a preexisting oliguria, as in hysteria. 

Specific Gravity. — The specific gravity of normal urine varies 
between 1.015 and 1.025, corresponding to 1200 to 1500 c.c, viz., the 
normal amount of urine voided in twenty-four hours. Pathologically, 
a specific gravity of 1.002 on the one hand and 1.060 on the other may 
occur, depending upon the amount of solids and fluids present, increas- 
ing as the solids increase, the amount of urine remaining the same, 
and decreasing as the amount of fluid increases, the solids remaining 
the same. The specific gravity is thus an index in a general way of 
the metabolic processes taking place in the body. 

The necessity of determining the specific gravity of the total 
amount of urine voided in a given case, and not that of an individual 
specimen passed during the twenty-four hours, becomes apparent 
upon considering the variations which may occur in the quantity of 
solids and liquids ingested during the day. The ingestion of large 
amounts of fluid would, of course, result in the passage of a 
correspondingly large quantity of urine within the next few hours, 
containing but a small amount of solids, and hence presenting a low 
specific gravity. From such an observation it would be erroneous 
to infer a diminished excretion of solids for the day, as succeeding 
specimens would in all probability be passed presenting a higher 
specific gravity. An observation made upon a specimen taken 
from the collected urine of the twenty-four hours moreover, can 
only then convey a correct idea if the total quantity is known. 

From the specific gravity the amount of solids can be calculated 
with sufficient accuracy for clinical purposes by multiplying the last 
two decimal points by 2, the number obtained indicating the amount 
of solids in 1000 c.c. of urine. 

From the rule, that the specific gravity of a urine is inversely pro- 
portionate to the amount of fluid eliminated, it must follow that 
whatever causes produce oliguria will also produce a high specific 
gravity, while all those causes which produce polyuria will similarly 
produce a low specific gravity, with the following exceptions: 

1. A diminished amount of urine with a lowered specific gravity 
occurs in many chronic diseases and toward the fatal termination of 
acute diseases, indicating a defective elimination of solids. 

2. The same may be observed in certain cases of edema. 

3. Following copious diarrhea, vomiting, and sweating. 

4. A high specific gravity is associated with polyuria in diabetes 
mellitus. 

Unfortunately the determination of the specific gravity and the 
solids contained in urine does riot furnish as valuable information 



GENERAL CHARACTERISTICS OF THE URINE 



367 



in many cases as would be expected a priori. This is largely owing 
to the fact that the organic constituents of the urine have a lower 
specific gravity than the inorganic salts, and especially the chlorides, 
which are usually present in considerable amount. It thus not infre- 
quently happens that the nitrogenous constituents are considerably 
increased, while the specific gravity is relatively low, owing to the 
absence or a diminution in the amount of chlorides. In other words, 





Fig. 132. — The pyknometer. 

while the specific gravity may be regarded 
as a fair index of the total amount of 
solids excreted, its increase or decrease 
furnishes no information as to the nature 
of the constituents causing such a change. 
Determination of Specific Gravity. 
— The specific gravity of the urine is most 
conveniently determined by means of a 
hydrometer indicating degrees varying 
from 1.002 to 1.040. Such instruments, 
constructed especially for the examina- 
tion of urine, are termed urinometers 
(Fig. 131). A good instrument should 
have a stem upon which the individual 
divisions are at least 1.5 mm. apart, and each division should corre- 
spond to 0.5 degree. 

Urinometers may also be purchased which are provided with a 
thermometer. Every instrument should be carefully tested by com- 
parison with a standard hydrometer. 

In order to determine the specific gravity in a given case a cylindrical 
vessel is nearly filled with urine and the urinometer slowly introduced. 



Fig. 131. — Urinometer. 



368 THE URINE 

the reading being taken at the lower meniscus as soon as the 
instrument has come to rest. 

Precautions: 1. The urinometer must be given ample room, and 
the reading should never be taken when the instrument touches the 
sides of the vessel, as owing to capillary attraction it is otherwise 
raised, causing the reading to be too high. 

2. The instrument must be perfectly dry and clean before being 
used, and should never be allowed to "drop" into the urine, as other- 
wise the weight of the instrument is increased by adhering drops of 
fluid, and the reading is too low. 

3. Any foam upon the surface of the urine should first be removed 
by means of a piece of filter paper, as it interferes with the accuracy 
of the reading; bubbles of air adhering to the instrument, and thereby 
elevating it, should be removed with a feather. 

4. The specific gravity should always be determined in specimens 
taken from the twenty-four-hour urine. 

5. If the quantity of urine is too small to determine its specific 
gravity with a urinometer, the following method may be employed: 

About 50 c.c. of urine are measured into a small bottle provided 
with a ground-glass stopper, or into a pyknometer like the one pic- 
tured in Fig. 132, and accurately weighed. The weight of the urine 
divided by its volume gives the specific gravity, which must, however, 
be corrected for the temperature of the urine. If accuracy is re- 
quired, such corrections should be made in every case, as the specific 
gravity increases or decreases by 1° for every 3° C. above or below 
the point for which the instrument is registered, viz., 15° C. 

Determination of the Solid Constituents. — As indicated above, the 
amount of solids can be calculated with a degree of accuracy sufficient 
for clinical purposes by multiplying the last two figures of the specific 
gravity by 2; the number obtained indicates the amount of solids in 
every 1000 c.c. of urine. If greater accuracy is required, the follow- 
ing method may be employed: 5 c.c. of urine, accurately measured, 
are placed in a watch-crystal containing a little dry sand (sand and 
crystal having been previously weighed) ; this is placed over a dish con- 
taining concentrated sulphuric acid, and under the receiver of an air 
pump which has been made perfectly air-tight by thoroughly lubricat- 
ing the ground-glass edge of the bell with mutton tallow and applying 
the bell with a slightly grinding movement to the ground-glass plate. 
The receiver is now exhausted and the urine allowed to remain in 
the vacuum for twenty-four hours, when the bell is again exhausted 
and left for twenty-four hours longer; at the end of this time the 
crystal is weighed, the difference between the two weights obtained 
indicating the amount of solids in 5 c.c. of urine, from which the 
percentage and total amount are readily calculated. 

The slight loss of ammonia which results when this method is 
employed scarcely affects the accuracy of the result. 



GENERAL CHARACTERISTICS OF THE URINE 369 

Reaction. — The reaction of the twenty-four-hour urine is, as a rule, 
acid; individual specimens, passed in the course of the same twenty- 
four hours, may be either alkaline, acid, or amphoteric. 

It has been generally held in the past that the acid reaction of 
normal urine is due to the presence of diacid phosphates. But it 
was assumed also that monosodium phosphate was present at the same 
time. Folin 1 has shown that this assumption is not correct, that the 
phosphates in clear urine are all of the monobasic kind, and that the 
acidity of such urines is ordinarily greater than the acidity of all the 
phosphates, the excess being due to free organic acids. 

An alkaline urine results when the alkalies exceed the acid equiva- 
lents in amount. This may occur under normal conditions (see below), 
and is then due to a preponderance of monacid over diacid phos- 
phates. An amphoteric urine (red litmus turned blue and blue litmus 
red) is the outcome, when the acid equivalents of diacid phosphates 
equal the basic equivalents of the monophosphates; this is essentially 
an accidental occurrence. 

As the alkalinity of the blood increases the acidity of the urine 
decreases, until an alkaline urine results. The degree of the alkalinity 
of the blood, however, depends essentially upon the nature of the 
food and the secretion of the gastric juice, viz., the hydrochloric acid. 
The ingestion of vegetable food, rich in salts of organic acids, which 
become oxidized in the body to the carbonates of the alkalies, will 
result in the passage of an alkaline urine, for the alkalies thus formed 
when absorbed into the blood are more than sufficient to neutralize 
completely all the acids present, and the elimination of neutral 
sodium phosphate alone takes place. In the case of animal food the 
reverse holds good. The alkaline carbonates here formed are not 
sufficient to neutralize the excess of acids, and diacid phosphate of 
sodium is hence eliminated in large quantity. 2 

As the alkalinity of the blood is increased during the secretion 
of the acid gastric juice, it may frequently happen, especially follow- 
ing the ingestion of a large amount of food, that an alkaline urine 
is voided. If this does not take place, the acidity of the urine is at 
least diminished, but increases again during the process of resorp- 
tion. 3 

If an acid urine is allowed to stand exposed to the air for a cer- 
tain length of time, its degree of acidity gradually diminishes and 
the reaction finally becomes alkaline. At the same time the urine 
becomes cloudy and deposits a sediment, which consists of ammonio- 
magnesium phosphate, MgNH 4 P0 4 + 6H 2 0, neutral calcium phos- 
phate, Ca 3 (P0 4 ) 2 , and still later contains ammonium urate, C 5 H 2 - 
(NH 4 ) 2 N 4 3 , in addition to the constituents of the primitive nubecula 

1 Amer. Jour, of Physiol., Feb., 1905, vol. xiii. 

2 E. Salkowski u. J. Munk, Virchow's Archiv, 1877, vol. lxxvi, p. 500. 

3 Quincke, Zeit. f. klin. Med., vol. vii. 

24 



370 THE URINE 

— i. e., a few mucous corpuscles and pavement epithelial cells. The 
entire volume of urine, moreover, remains cloudy, owing to the 
presence of innumerable bacteria. The odor becomes extremely 
disagreeable and distinctly "urinous." In short, "ammoniacal de- 
composition" has occurred. This has been shown to depend upon 
the action of certain bacteria, notably the Micrococcus urese and the 
Bacterium urese, which are present in the air. 1 These organisms cause 
the decomposition of the urea found in every urine, with the forma- 
tion of ammonium carbonate, according to the following equations: 

CO(NH 2 ) 2 +2H 2 = (NH 4 ) 2 C0 3 
(NH 4 ) 2 C0 3 = 2NH 3 +H,.0 +C0 2 . 

An alkaline urine, the alkalinity of which is not due to ammo- 
niacal fermentation, however, but to other causes, as indicated 
above, may, of course, undergo the same change as an acid urine; 
but it is necessary to distinguish sharply between these two varieties 
of alkaline urines, as the recognition of the cause of the alkalinity is 
very often most important in diagnosis. The distinction is readily 
made by fastening a piece of sensitive red litmus paper in the cork 
of the bottle containing the urine. If the alkalinity of the urine is 
due to the presence of ammonia, the litmus paper will turn blue, but 
soon changes to red when exposed to the air; while a urine the 
alkalinity of which is due to the presence of fixed alkalies will turn 
red litmus paper blue only when immersed in the urine, the change 
in color at the same time persisting. 

As ammoniacal decomposition can also occur within the urinary 
passages, it is important, whenever an alkaline reaction due to the 
presence of ammonia is observed, to test the urine at once upon being 
voided, or, still better, to procure a portion with a catheter. Such 
urines are frequently seen in cases of cystitis the result of paralysis, 
urethral stricture, gonorrhea, etc. In this connection it is interest- 
ing to note that whereas in old, neglected cases of cystitis an alkaline 
reaction is frequently observed, Brown has shown that in the great 
majority of cases of cystitis, both acute and chronic, and also in those 
of pyelitis and pyelonephritis, the urine is acid. 2 

An intensely acid reaction is observed in almost all concentrated 
urines, especially in fevers, in certain diseases of the stomach asso- 
ciated with a diminished or suspended secretion of hydrochloric acid, 
in gout, lithiasis, acute articular rheumatism, chronic Bright's dis- 
ease, diabetes, leukemia, scurvy, etc.. Whenever a very acid urine 
is secreted for a considerable length of time, the possibility of renal 
irritation and the formation of concretions should be borne in mind. 

An alkaline urine the alkalinity of which is not owing to the pres- 

u W. Leube, "Ueber die ammoniakalische Harngahrung," Virchow's Archiv, 
1885, vol. c, p. 555. 

2 T, R. Brown, Johns Hopkins Hosp. Rep., 1901, vol. x, p. 11. 



GENERAL CHARACTERISTICS OF THE URINE 371 

ence of ammonia, but to fixed alkali, is observed in certain cases 
of debility, especially in the various forms of anemia, following the 
resorption of alkaline transudates, the transfusion of blood, frequent 
vomiting, a prolonged cold bath, etc. It may also be due to the 
ingestion of certain drugs, viz., salts of the organc acids and alkaline 
carbonates, the former being transformed into the latter, as has been 
mentioned. An increase in the degree of acidity may similarly take 
place after the ingestion of mineral acids. 

Of interest is the observation of Pick 1 that in twenty-four to forty- 
eight hours after the crisis in pneumonia the urine shows a marked 
decrease in its acidity, becoming neutral or even alkaline. This 
phenomenon, which was observed in 31 out of 38 cases, persists 
for a day or a day and a half, and then the acidity returns. In all 
likelihood the change is due to absorption of the large amounts of 
sodium which are present in the exudate. 

An increase in the acidity of the urine upon standing has repeat- 
edly been observed, and is probably due to the formation of new 
acids from preexisting acid-yielding substances, such as certain 
carbohydrates, alcohol, etc., which have undergone fermentation. 
This phenomenon is frequently observed in diabetic patients. 

A decrease in the acidity of normal urine upon standing, however, 
is the rule, owing to a gradual decomposition of sodium urate by 
the acid sodium phosphate, acid sodium urate, and, later on, uric 
acid resulting, which are thrown down as a sediment in consequence 
of the diminished acidity of the urine, and which, hence, no longer 
influence its reaction. This is shown in the equations: 

1. NaH 2 P0 4 +C 5 H 2 Na 2 N 4 3 = Na 2 HP0 4 +C 5 H 3 NaN 4 3 . 

2. NaH 2 P0 4 +C 5 H 3 Na N 4 3 = Na 2 HP0 4 +C 5 H 1 N 4 > 

Determination of the Acidity of the Urine.— Folin has shown that 
the methods of Freund, Lieblein and Nageli, which have heretofore 
been largely in use, are inapplicable and has suggested the following 
procedure : 

Folin's Method. — The total acidity which indicates the acidity due 
to diacid phosphates and free organic acids is first determined as 
follows: 25 c.c. of urine are treated with 1 or at most 2 drops of \ 
per cent, alcoholic solution of phenolphthalein and 15 to 20 grams of 
powdered potassium oxalate. The solution is shaken for about a 
minute and titrated at once with decinormal sodium hydrate solution 
until a faint, yet distinct pink color is obtained. The flask should be 
shaken during the titration, so as to keep the solution as strong as 
possible in oxalate. The acidity is expressed in terms of decinormal 
sodium hydrate solution for the total amount of urine of twenty-four 
hours. The total acidity is termed T. 

1 "The Urine in Pneumonia," Munch, med. Woch., 1898, No. 17. 



372 THE URINE 

In a second specimen the total phosphates are then determined, 
the value being termed P (see Phosphates). The result is expressed in 
terms of decinormal acid, viz., alkali as above (1 c.c. y ¥ = 7.1 mgrms. 
of P 2 5 ). T minus P then indicates the acidity due to uncombined 
organic acids (O. A.), and the difference the mineral acidity (M. A.). 

It may happen that the acidity calculated from the total phosphates 
is greater than the titrated acidity; in that case practically no free 
organic acids are present and the titrated acidity represents the 
amount of phosphates present in the diacid form. Urines of this kind 
are turbid, unless they are also free from calcium (Folin). 

As average normal value for the acidities of the total bulk of twenty- 
four hours' urine Folin obtained 617 (c.c. y 1 -^ n. acid, viz., alkali), of 
which 304 was referable to mineral and 313 to organic acidity. The 
corresponding minimal and maximal values were T 554, viz., 669; 
M. A. 204, viz., 417; O. A. 252, viz., 378. 

With this method a complete revision of all the work previously 
done will be necessary. The older results given above have reference 
only to the old method of titration with a one-tenth normal solution of 
sodium hydrate. 

Literature. — Folin, Amer. Jour, of Physiol., 1903, vol. ix, p. 265; and ibid., 
1905, Feb., pp. 53 and 54, and ibid., p. 102. 

Determination of the Mineral Acidity or the Excess of Mineral 
Acids or Bases. — Folin's method may be employed instead of 
determining all the different metals and acids separately as Bunge, 
Magnus Levy and others have done. 

To 25 c.c. of urine in a platinum dish is added from 0.3 to 0.5 
gram of potassium carbonate, weighed within an accuracy of two- 
tenths of a mgrm. The solution is evaporated to dryness, and the 
residue ignited, when perfectly dry, over a radial burner, using at first 
a very low heat, and at no time allowing the dish to become more 
than faintly red hot. The dish is heated at this temperature for one 
hour, then cooled, when 10 c.c. of hydrogen peroxide are added and 
evaporated. The dried residue is ignited as before for one hour. 
It is dissolved in an excess of tenth normal hydrochloric acid and water 
(50 to 75 c.c. yo HC1), transferred to an Erlenmeyer flask, boiled 
to remove carbonic acid, and cooled. One or two drops of phenol- 
phthalein solution and a few crystals of neutral potassium oxalate 
(to precipitate the calcium) are added, and the solution titrated as 
usual. The ammonia, the acidity of the hydrogen peroxide, and the 
acidity of the organic sulphur (neutral and ethereal, 8 grams of which 
are taken to represent 1 c.c. tenth normal acid) must be subtracted 
from the result given by the direct titration. These values, as well 
as the acidimetric value of the potassium carbonate, must be separately 
determined. 

This procedure gives very reliable results, if proper care is used 



CHEMISTRY OF THE URINE 373 

in the evaporation and the burning of the urine. It is to be used only 
when the actual excess of mineral acids above that necessary for 
the neutralization of the mineral bases is to be estimated, or when 
the total amount of organic acids in urine (whether free or combined 
with bases) is to be determined (Folin). 



CHEMISTRY OF THE URINE. 

General Chemical Composition of the Urine.— A general idea of 
the chemical composition of the urine and the quantitative variations 
of the individual components may be formed from the following 
table, which I have constructed from analyses made in my labo- 
ratory. The individuals from which the urines were obtained were 
adults, and their general mode of life, as regards diet, exercise, etc., 
was that of the average American city dweller. In addition, the 
following substances may be encountered under pathological con- 
ditions: serum albumin, serum globulin, albumoses, mucin (nucleo- 
albumin), glucose, lactose, inosit, dextrin, biliary constituents, viz., 
bile acids and bile pigments, blood pigments, melanin, leucin, tyro- 
sin, oxybutyric acid, allantoin, fat, lecithin, cholesterin, acetone, 
alcohol, Baumstark's substance, urocaninic acid, cystin, hydrogen 
sulphide, and still others. 

Analysis of Urine. 

Water 1200-1700 grams. 

Solids 60.0 

Inorganic solids 25.0-26.0 " 

Sulphuric acid (HoS0 4 ) 2.0-2.5 " 

Phosphoric acid (PoO,) 2.5-3.5 " 

Chlorine (NaCl) . 10.0-15.0 " 

Potassium (K,0) 3.3 

Calcium (CaO) ......... 0.2-0.4 

Magnesium (MgO) 0.5 " 

Ammonia (NH 3 ) .0.7 " 

Fluorides, nitrates, etc. . . . . . . 0.2 

Organic solids 20.0-35.0 ■" 

Urea 20.0-30.0 " 

Uric acid 0.2-1.0 " 

Xanthin bases . . . 1.0 

Kreatinin . 0.05-0.08 " 

Oxalic acid . 05 

Conjugate sulphates 0.12-0.25 " 

Hippuric acid 0.65-0.7 " 

Volatile fatty acid 0.05 

Other organic solids . . 2.5 " 

Quantitative Estimation of the Mineral Ash of the Urine. — In 
order to estimate the amount of mineral ash in the urine the follow- 
ing method may be employed : 50 c.c. of urine are evaporated to dryness 
in a weighed porcelain dish, at a temperature of 100° C, and then 



374 



THE URINE 



heated, while covered, over the free flame until gases cease to be 
evolved, care being taken not to heat too strongly in order to avoid 
sputtering. The residue is taken up with distilled boiling water, 
and, after standing, filtered through a Schleicher and Schiill filter, 
the weight of the ash of which is known. The dish and the contents 
of the filter are well washed with hot water. Filtrate and wash- 
ings are set aside and the dish and 
filter dried in the oven at 115° C. 
The filter is now placed in the 
dish and slowly incinerated. So 
soon as the ash has turned white 
the filtrate and washings are placed 
in the same dish, evaporated at 
100° C, and then carefully heated 
over the free flame. Upon cool- 
ing in the desiccator (Fig. 133) the 
dish with its contents is weighed, 
the difference between its present 
and previous weight indicating the 
quantity of ash contained in 50 
c.c. of urine. 

Precautions: 1. Care should be taken to allow the dish to become 
faintly red only for a moment, as some of the chlorine is otherwise 
volatilized. Some phosphoric acid may also escape, and too strong 
a heat, moreover, may cause the transformation of sulphates into 
sulphides, the organic material present acting as a re ducing agent. 
2. If the organic ash is not completely incinerated, it is best to 
allow the dish to cool and then to moisten the ash with a few drops 
of dilute sulphuric acid, when the heating is continued. 




Fig. 133. — Desiccator. 



The Chlorides. 



The chlorides which are excreted in the urine are derived from the 
food. As they are thus present in a much larger amount than all 
other inorganic salts combined, and in quantity more than sufficient 
to supply the needs of the body economy, the relatively large amount 
of chlorides found in the urine under physiological conditions, as 
compared with the other inorganic constituents, is readily explained. 

Of the alkalies in the urine, sodium in combination with chlorine 
exists in greatest amount, and for clinical purposes it is most con- 
venient to calculate the total quantity of chlorides in terms of sodium 
chloride; a small proportion also occurs combined with potassium, 
ammonium, calcium, and magnesium. 

From 11 to 15 grams of sodium chloride, representing the total 



CHEMISTRY OF THE URINE 375 

quantity of chlorine, are normally eliminated in the twenty-four 
hours, the amount depending, of course, directly upon that contained 
in the food ingested. If the amount of nourishment is diminished, a 
decrease in the elimination of the chlorides is observed. If this is 
carried to the point of starvation, the chlorides disappear almost 
entirely from the urine, the traces remaining being derived from the 
body fluids. The latter retain tenaciously a certain amount, which 
differs but slightly from that normally present. If at this stage food 
containing sodium chloride is again taken, a portion will be retained 
in the body until the original equilibrium is restored. A similar 
retention may be observed for a few days following the ingestion of 
large quantities of water, which causes an increased elimination of 
chlorides. * 

This tenacity on the part of the body in retaining sodium chloride 
is strikingly seen when the potassium salt is substituted for the 
sodium salt; in this case the amount of the sodium in the serum 
of the blood will be found to vary very slightly. 

It has also been shown that the excretion of sodium chloride 
can be increased very materially by the ingestion of potassium 
salts, notably the neutral potassium phosphate (K 2 HP0 4 ). This is 
supposed to decompose the sodium chloride present in the serum, 
with the formation of potassium chloride and neutral sodium 
phosphate, which are both eliminated as foreign material; a point 
is finally reached, however, when the sodium chloride ceases to be 
excreted. 

This provision of the economy, in virtue of which an increase in 
the elimination of the salt is followed by its retention, and a pre- 
vious retention by an increased elimination, is supposed to be inti- 
mately associated with the albuminous metabolism of the body. It 
may be stated, as a general rule, that any increase in the amount of 
circulating albumin will be followed by an increased elimination of 
chlorides, these having been previously retained by the albuminous 
bodies in consequence of the great affinity which exists between 
them. At the same time the elimination of the chlorides is influ- 
enced by the quantity of urine excreted, increasing and decreasing 
with its volume. 

Pathologically the excretion of the chlorides may vary within 
wide limits, diminishing on the one hand to zero and increasing 
on the other to 50 grams or more in the twenty-four hours. A 
marked diminution, which in some cases may go on to a total absence, 
was formerly thought to be pathognomonic of acute croupous pneu- 
monia. 1 More modern investigations, however, have shown that 
such a condition occurs to a greater or less degree in most acute 

1 Rettenbacher, Wien. med. Zeit., 1850, p. 373. Heller, Heller's Archiv, 1844, 
vol. i, p. 23. 



376 THE URINE 

febrile diseases, such as scarlatina, roseola, variola, typhus and 
typhoid fevers, recurrens, and acute yellow atrophy. Intermittent 
fever appears to be an exception to this rule; usually it is true the 
chlorides are diminished, but not to the extent seen in the other 
diseases mentioned. They have, moreover, been found to increase 
during and sometimes immediately after a paroxysm, this increase 
being, of course, followed by a corresponding diminution. 

The chlorides are diminished in all acute and chronic renal dis- 
eases associated with albuminuria. 1 In this connection it is interest- 
ing to note that in cases of nephritis associated with edema and other 
transudates the withdrawal of the chlorides from the food results in 
marked improvement and in some cases in the complete disappear- 
ance of the effusion. 

In all cases of carcinoma of the stomach, and in chronic hyper- 
secretion associated with dilatation, a decrease is observed, which in 
certain cases of hypersecretion and hyperacidity, the result of gastric 
ulcer, may go on to a total absence. 2 

In anemic conditions the chlorides are likewise diminished, as 
also in rickets. In melancholia and idiocy a striking decrease is 
observed; in dementia, chorea, and pseudohypertrophic paralysis 
this is less marked. 

A total absence has been noted in pemphigus foliaceus, and a con- 
siderable diminution in the beginning of impetigo, as also in chronic 
lead poisoning. 

The chlorides are found in increased amount in all conditions in 
which retention has previously occurred, chief among these being the 
acute febrile diseases and cases in which a resorption of exudates and 
transudates, associated with an increased diuresis, is taking place. 
A marked increase has been noted in some cases of diabetes insipidus, 
in which 29 grams have been eliminated in the twenty-four hours. 3 
A similar increase may occur in prurigo, in which, in one instance, 
29.6 grams were passed in twenty-four hours. 4 In cases of general 
paresis, during the first stage, an increased elimination goes hand in 
hand with an increased ingestion of food. In epilepsy the polyuria 
following the attacks is associated with an increase in the chlorides, 

Of drugs, certain diuretics, and some of the potassium salts, as 
has been mentioned, produce an increase: the chlorine contained in 
chloroform, whether administered internally, or as an anesthetic, is 
in part excreted in the form of a chloride. Salicylic acid, on the 
other hand, is said to cause a temporary diminution. 

It is of practical importance to note that in acute febrile diseases 
the diminution in the chlorides appears to vary with the intensity 

1 Rohmann, Zeit. f. klin. Med., 1886, vol. i, p. 513. 

2 Gluzinski, Berlin, med. Woch., 1887, vol. xxiv, 983. 

3 Oppenheim, Zeit. f. klin. Med., vol. vi. 

4 v. Brueff, Wien. med. Woch., 1871, p. 552. 



CHEMISTRY OF THE URINE 377 

of the disease, a decrease to 0.05 gram pro die justifying the con- 
clusion that the case under oservation is of extreme gravity. It 
may at times also indicate a preceding attack of severe diarrhea 
or the formation of exudates of considerable extent. A continued 
increase, on the other hand, should lead to the conclusion that the 
patient's condition is improving. 

The elimination of the chlorides also furnishes a fair index to the 
digestive powers of the patient. All other causes which might lead 
to an increase or decrease being eliminated, an excretion of from 10 
to 15 grams indicates a fair condition of the appetite and a normal 
digestive power, a decrease being associated with the reverse. 

An increased elimination of chlorides occurring in cases of edema, 
and associated with the existence of serous exudates, is always of 
good prognostic omen, pointing to a resorption of the fluid. 

A continued elimination of more than 15 to 20 grams, all other 
causes being excluded, may be considered as pathognomonic of dia- 
betes insipidus. 

Of late attention has been directed to the ratio between the elimi- 
nation of the chlorides and the total nitrogen. With an ordinary 
diet this is as 1 to 1 (Salkowski), even though the total amount of chlo- 
rides may not amount to 10 to 15 grams, but may be as low as 7 to 10 
grams. In disease this ratio may be much disturbed owing to chlo- 
ride retention (1 CI to 15 N); a change toward the normal is cceteris 
paribus a favorable sign. 

Test for Chlorides in the Urine. —The recognition of the chlorides 
in the urine is based upon the fact that silver nitrate causes their 
precipitation. The sliver chloride thus formed is insoluble in nitric 
acid. 

The test is made in the following manner: A few cubic centimeters 
of urine are acidified in a test-tube with about 10 drops of pure nitric 
acid, and treated with a few cubic centimeters of silver nitrate solu- 
tion (1 to 20). The occurrence of a white precipitate indicates the 
presence of chlorides. An idea may be formed at the same time of 
the quantity present; the occurrence of a heavy, caseous precipitate 
points to a large amount. Albumin, if present, must first be removed 
by boiling, after acidifying the urine with a few drops of dilute acetic 
acid. 

Quantitative Estimation of the Chlorides by the Method of 
Salkowski-Volhard. 1 — When a solution of silver nitrate acidified with 
nitric acid is treated with a solution of potassium sulphocyanide or 
ammonium sulphocyanide, in the presence of a ferric salt, the 
potassium sulphocyanide first causes the precipitation of white silver 
sulphocyanide, which, like silver chloride, is insoluble in nitric acid. 
As soon as every trace of silver is precipitated, it combines with 

1 E. Salkowski, Zeit. f. physiol. Chem.. vol. i, p. 16, and vol. ii, p. 379. 



378 THE URINE 

the ferric salt to form ferric sulphocyanide, which is of a blood-red 
color. If the potassium sulphocyanide solution is of known strength, 
it is possible to estimate accurately the amount of silver present in 
the solution, the ferric salt serving as an indicator of the end of the 
reaction between the silver and the potassium sulphocyanide. 

Application to the urine: to urine which has been acidified with 
nitric acid an excess of a silver solution of known strength is added, 
and the silver not used in the precipitation of the chlorides then esti- 
mated as indicated above. The difference between the quantity thus 
found and the total amount used will be that consumed in the pre- 
cipitation of the chlorides, from which, knowing the strength of the 
silver soluion, its equivalent in terms of sodium chloride is readily 
determined. 

Reagents required: 

1. A solution of silver nitrate of such strength that each cubic 
centimeter shall correspond to 0.01 gram of sodium chloride. 

2. A solution of potassium sulphocyanide of such strength that 
25 c.c. shall correspond to 10 c.c. of the silver nitrate solution. 

3. A solution of a ferric salt, such as ammonioferric alum, satu- 
rated at ordinary temperature. 

4. Nitric acid (specific gravity 1.2). 
Preparation of these solutions: 

1, As pointed out, the silver nitrate solution is made of such 
strength that each cubic centimeter shall correspond to 0.01 gram 
of sodium chloride. 

The silver nitrate must be pure, and it is best to use the crystal- 
lized salt, and not the sticks wrapped in paper, which always contain 
reduced silver. In order to test the purity of the salt, about 1 gram 
is dissolved in distilled water, heated to the boiling point, the silver 
precipitated by dilute hydrochloric acid and filtered off. When 
evaporated in a platinum crucible the filtrate should leave either no 
residue at all or only a very faint one; otherwise it is necessary to 
recrystallize the salt until the desired degree of purity is reached. 

The determination of the quantity to be dissolved in 1000 c.c. of 
water is based upon the fact that 1 molecule of silver nitrate (mole- 
cular weight 170) combines with 1 molecule of sodium chloride 
(molecular weight 58.5) to form silver chloride and sodium nitrate. 
As the solution of silver nitrate shall be of such strength that 1 c.c. 
corresponds to 0.01 gram of sodium chloride, or 1000 c.c. to 10 grams, 
the quantity to be dissolved in 1000 c.c. is found according to the 
following equation: 

58.5: 170: : 10 s, 58.5.x = 1700, x = 29.059. 

Theoretically, then, this quantity should be dissolved in 1000 c.c. 
of water. It is better, however, to dissolve it in a quantity some- 
what less than 1000 c.c, such as 900 or 950 c.c, as the silver salt 



CHEMISTRY OF THE URINE 379 

contains water of crystallization and the weighed-off quantity would 
not represent the exact amount required, but less, the correcting of 
a solution which is too strong being a much simpler matter than 
that of a solution which is too weak. 

To make this correction, or, in other words, to bring the solution 
to its proper strength, 0.15 gram of sodium chloride, which has 
previously been dried carefully by heating in a platinum crucible, is 
accurately weighed off, dissolved in a little distilled water, and further 
diluted to about 100 c.c. To this solution a few drops of a solution 
of potassium chromate are added, when the mixture is titrated with 
the silver solution. The silver nitrate will first precipitate the sodium 
chloride, and then combine with the potassium chromate, forming red 
silver chromate. The slightest orange tint remaining after stirring 
indicates the end of the reaction. Were the solution of the silver 
nitrate of the proper strength, exactly 15 c.c. should have been used, 
as each cubic centimeter shall represent 0.01 gram of sodium chloride. 
As a matter of fact, less will in all probability be needed, the solution 
having been purposely made too strong. Its correction then becomes 
a simple matter, as it is, merely necessary to determine the degree of 
dilution required. 

Supposing that 29.059 grams of silver nitrate were dissolved in 
900 c.c. of water, and that 14.5 c.c. instead of 15 c.c. had been re- 
quired to precipitate the 0.15 gram of sodium chloride, it is evident 
that each 14.5 c.c. of the remaining solution must be diluted with 
0.5 c.c. of water. It is, hence, only necessary to divide the number of 
cubic centimeters of the silver nitrate solution remaining by 14.5; 
the result multiplied by 0.5 represents the amount of water which 
must be added in order to bring the solution to the required strength. 
Hence the rule for the correction of a solution which has been found 
too strong: 

N.d 

C= 7 

n 

in which & represents the number of cubic centimeters of water 
which must be added to the solution remaining; N the total number 
of cubic centimeters remaining after titration; n the number of 
cubic centimeters consumed in one titration; and d the difference 
between the number of cubic centimeters theoretically required and 
that actually used in one titration. 

In the example given the equation would then read: 

936.5 X 0.5 
C-— e^—- 32.29. 

32.29 c.c. of distilled water are added to the remaining 936.5 c.c, 
when the strength of the solution is tested by a second titration. If 
the solution is found too weak, it is best to make it too strong, and 
then to correct as described. 



380 THE URINE 

2. Preparation of the potassium sulphocyanide solution: as 
1 molecule of silver nitrate (molecular weight 170) combines with 
1 molecule of potassium sulphocyanide (molecular weight 97), the 
quantity of the latter to be dissolved in 1000 c.c. of water is 
found from the following equation: 

170: 97: : 11,6236: x; 170 x = 11.6236X97; x = 6.6. 

As potassium sulphocyanide is extremely hygroscopic, a solution 
is made which is too strong, by dissolving about 10 grams of the 
salt in 900 c.c. of distilled water. In order to bring this solution to 
its proper strength, 10 c.c. of the silver solution are diluted to 100 
c.c; 4 c.c. of nitric acid (specific gravity 1.2) and 5 c.c. of the am- 
monioferric alum solution are added, when the mixture is titrated 
with the potassium sulphocyanide solution; the end reaction is 
recognized by the production of a slightly reddish color, which per- 
sists on stirring. The sulphocyanide solution having been purposely 
made too strong, it will be found that less than 25 c.c. are needed to 
precipitate all the silver present. The quantity of water necessary 
for dilution is ascertained, as above, according to the formula 

°~ n 

3. The solution of ammonioferric alum is a solution saturated at 
ordinary temperatures, care being taken to ensure the absence of 
chlorides in the salt, which may be effected, if necessary, by recrys- 
tallization. 

Method as Applied to the Urine. — 10 c.c. of urine are placed in a 
small stoppered flask bearing a 100 c.c. mark, diluted with 50 c.c. 
of distilled water, and acidified with 4 c.c. of nitric acid. From a 
burette 15 c.c. of the standard solution of silver nitrate are added. 
The mixture is thoroughly agitated and diluted with distilled water 
to the 100 c.c. mark. The silver chloride formed is filtered off 
through a dry, folded filter into a dry graduate; 80 c.c. of the filtrate 
are placed in a beaker, and, after the addition of 5 c.c. of the ammonio- 
ferric alum solution, titrated with the sulphocyanide solution until 
the end reaction — i. e., a slightly reddish tinge — is seen. If necessary, 
two such titrations should be made, the sulphocyanide solution being 
added 1 c.c. at a time in the first, while in the second the total number 
of cubic centimeters needed to bring about the end reaction, less 1 
c.c, are added at once, and then 0.1 c.c. at a time. 

The amount of chlorides present in the urine is calculated as fol- 
lows : 

Example. — Total quantity of urine 600 c.c; 6.5 c.c of the sul- 
phocyanide solution were required to bring about the end reaction 
in 80 c.c of the filtrate; this would correspond to 8.125 c.c for the 



CHEMISTRY OF THE URINE 381 

total 100 c.c. of filtrate, representing 10 c.c. of urine, as is seen from 
the equation 

100 n 5 n 
n : 80 : : x : 100; 80 a: =100 n; x = -^- = ^' 

in which x represents the number of cubic centimeters correspond- 
ing to 100 c.c. of the filtrate, and n the number of cubic centimeters 
actually used. 

These 8.125 c.c. were used in precipitating the silver nitrate not 
decomposed by the chlorides. As 25 c.c. of the sulphocyanide solu- 
tion correspond to 10 c.c. of the silver solution, the excess of silver 
solution in cubic centimeters is found from the equation 

10 N 2A T 
25 : 10 : : N : x; 25x=10A; x = -^- = —^~ > 

in which x represents the excess of the silver solution in cubic centi- 
meters, and N that of the sulphocyanide solution as found according 
to the equation above, x in this case being 3.25 c.c. 

The difference between the total amount of silver solution em- 
ployed (i. e., 15 c.c.) and the excess (i. e., 3.25 c.c.) indicates the 
number of cubic centimeters necessary for the precipitation of the 
chlorides in 10 c.c. of urine. In the case under consideration 11.75 
c.c. were employed. As 1 c.c. of the silver solution represents 0.01 
gram of sodium chloride, there must have been present in the 10 c.c. 
of urine 0.1175 gram; in 100 c.c, hence, 1.175 grams, and in the 
total amount — i. e., 600 c.c. of urine — 7.05 grams. 

The method described may be employed in the presence of albu- 
mins, albumoses, and sugar; the urine, however, must be fresh, so 
as to ensure the absence of nitrous acid. 

Direct Method. 1 — If accuracy is not required, the following method 
may be employed: 10 c.c. of urine are diluted with distilled water 
to 100 c.c. and treated with a few drops of a solution of potassium 
chromate. This mixture is titrated with a one-tenth normal solution 
of silver nitrate until the end reaction is reached — i. e., a faint 
orange tinge — which no longer disappears on stirring. The number 
of cubic centimeters used multiplied by 0.01 will indicate the amount 
of chlorides present in 10 c.c. of urine. 

As uric acid, the xanthin bases, hyposulphites, sulphocyanides, 
and pigments are also precipitated by the silver nitrate, the end 
reaction is delayed; moreover, unless the urine is very pale, its recog- 
nition may be difficult, and the error thus caused considerable. This 
is especially true of febrile urines which contain only a small amount 
of chlorides. 

Should iodides or bromides have been taken, these must first be 
removed, as silver iodide and bromide, which are insoluble in nitric 
acid, would give too high a value. 

1 F. Mohr, Lehrbuch d. Titrirmethode, 1856, ii, p. 13. 



382 THE URINE 



The Phosphates. 

The phosphates occurring in the urine are sodium, potassium, 
calcium, and magnesium salts of the tribasic acid H 3 P0 4 . The most 
important of these, as was pointed out in the chapter on Reaction, 
is the diacid sodium phosphate NaH 2 P0 4 , to which the acidity of 
the urine is in part due. It is owing to the presence of this salt in 
the urine that the calcium phosphate is held in solution; the fact, at 
least, that calcium and magnesium phosphate are thrown down when 
the urine is neutralized would point to this conclusion. 

The composition of the phosphates is liable to considerable varia- 
tion, depending upon the degree of acidity of the urine. As would 
be expected, diacid sodium phosphate and diacid calcium phosphate 
are present in an acid urine; in an amphoteric urine, in addition to 
these there are found disodium phosphate, monocalcium phosphate, 
and monomagnesium phosphate, while in an alkaline urine trisodic 
phosphate, neutral calcium phosphate, and neutral magnesium phos- 
phate may be present. 

The alkaline phosphates normally exceed the earthy phosphates by 
one-third, and sodium is combined with by far the greater amount 
of phosphoric acid, the potassium salt normally occurring in only 
very small amounts. 

In addition to the mineral phosphates, phosphoric acid is excreted 
also in combination with glycerin as glycerin-phosphoric acid, which 
need not, however, be considered in a quantitative estimation, as it 
is present only in traces. 1 

As in the case of the chlorides, the greater portion of the phos- 
phates is derived from the food, while only a small portion is refer- 
able to the tissue proteids. But just as the percentage of sulphur 
varies in the different tissues, so also does that of phosphorus vary; 
nerve tissue, for example, which is very rich in lecithins and nucleins, 
yields relatively more phosphorus than muscle tissue. 

Not all the phosphoric acid ingested, however, is excreted in the 
urine, as one-third to one-fourth of the total quantity is eliminated 
in the feces. 

The quantity of phosphoric acid excreted, which normally varies 
between 2.5 and 3 grams, is thus largely dependent upon the amount 
ingested, increasing with an animal and decreasing with a vege- 
table diet. 2 During starvation a considerable increase is likewise 
observed, referable, no doubt, to an increased destruction of bony 
tissue, which is very rich in the phosphates of the alkaline earths. 
In accordance with this view, increased amounts of calcium and 

1 Lepine et Eymonnet, Comp.-rend. de la Soc. de biol., 1882. 

2 Zulzer, Virchow's Archiv, vol. lxvi, p. 223. 



CHEMISTRY OF THE URINE 383 

magnesium are also seen during starvation. The relation between 
the excretion of phosphoric acid and nitrogen, normally 1 to 7, changes, 
moreover, in such a manner that both the absolute and the relative 
amount of phosphoric acid, as compared with the nitrogen, increases; 
this leads to the conclusion that in addition to the muscles some 
other tissue rich in phosphorus and relatively poor in N must suffer 
during the process, and the only one which could enter into con- 
sideration is bone. 1 If at this time food containing phosphorus is 
again given, a retention will take place, so that the general rule stated 
in the chapter on Chlorides, that increased elimination is followed 
by a certain degree of retention, and that a previous retention is fol- 
lowed by an increased elimination, seems to hold good for all the 
mineral acids found in the urine (see also the chapter on Sulphates). 

An increased elimination is caused also by the ingestion of large 
quantities of water, which is followed by a certain degree of retention. 

Observations on the phosphatic excretion during muscular exercise 
have not given uniform results. 2 Mental exercise appears to cause a 
diminished excretion of the alkaline phosphates and an increased 
elimination of the earthy phosphates. 3 The latter also takes place 
during sleep. 

In disease the total amount of phosphates may either be increased 
or diminished. 

A diminished elimination is observed in most cases of acute febrile 
disease, such as pneumonia, typhoid fever, typhus fever, recurrens, 
during a paroxysm of intermittent fever, etc. The degree of dimi- 
nution is usually proportionate to the severity of the disease, reaching 
its lowest figure as death approaches. Such a state of affairs may, 
at first sight, appear paradoxical in view of what has been said above 
of the effects of tissue destruction upon the elimination of phos- 
phates. It is necessary, however, to distinguish sharply between an 
increased production and an increased elimination; in all probability 
a retention occurs analogous to that of the chlorides, which may be 
observed under the same conditions. It has been supposed that the 
phosphates set free during the process of tissue destruction are 
utilized in the building up of new leukocytes, and an increase in 
these is actually noted in some of the diseases mentioned. A dimin- 
ished excretion of phosphates is, however, not always observed, 
and an increased elimination may occur in certain cases. In fatal 
cases this condition may persist even until the time of death. It 
is very difficult to give a satisfactory explanation of this fact at the 
present time. The phenomenon, in typhoid fever at least, appears 
to be connected with the intensity of the nervous manifestations, and 
Robin concludes that here an increased elimination during the fastig- 

1 Ziilzer, loc. cit. 

2 Fleischer u. Penzoldt, Virchow's Archiv, vol. lxxxvii, p. 210. 

3 Mariet, Compt.-rend. de la Soc. de biol., 1884. 



384 THE URINE 

ium is an unfavorable omen, while an increase during defervescence 
warrants a favorable prognosis. A similar decrease in the phosphates 
has also been observed in pulmonary phthisis associated with high 
fever. 1 

Very interesting and important is the diminshed excretion of 
phosphates associated with acute and, to some extent also, with 
chronic nephritis, amyloid degeneration of the kidneys, and the 
anemias, in which an actual insufficiency on the part of the kidneys 
in the elimination of these salts appears to exist. 2 

A diminished or, at least, no increased excretion is usually seen in 
certain diseases of the bones, such as osteomalacia. This may depend 
either upon a retention or an elimination through other channels. 
The earthy phosphates especially are found in greatly diminished 
amount, or may even be absent altogether in certain cases of 
nephritis. A similar condition is observed in acute and chronic 
rheumatism. 

The data regarding the phosphatic elimination in nervous and 
mental diseases are, on the whole, scanty and by no means uniform. 

During attacks of hysteria majoi, in contradistinction to epilepsy, 
in which an increased elimination takes place, the phosphates are 
diminished, the degree of diminution being generally proportionate 
to the intensity of the attack, increasing again together with the other 
urinary constituents with the subsequent increase in the diuresis. 3 

In chronic lead poisoning a diminution to one-third of the normal 
quantity may occur. Very low figures have been noted in Addison's 
disease, in acute yellow atrophy (in which even a total absence may 
occur), and in certain cases of hepatic cirrhosis. In gout the phos- 
phoric acid curve follows that of the uric acid quite closely, decreas- 
ing before the onset of the acute symptoms and then rising and 
reaching its maximum about the third day (see Uric Acid). 4 

An increased elimination of phosphates, on the other hand, amount- 
ing in some cases to 7 or even to 9 grams in the twenty-four hours, 
has been described by Teissier, of Lyon, under the name of phos- 
phatic diabetes, the patient presenting various symptoms commonly 
seen in diabetes mellitus ; sugar, however, is usually absent. Whether 
or not phosphatic diabetes is a disease sui generis is not certain. 5 

In true diabetes mellitus a curious relation has been found to 
exist between the elimination of sugar and of phosphates, the quan- 
tity of the latter rising and falling in an inverse ratio to the amount 
of sugar. In diabetes insipidus a slight increase is at times found. 

1 Edlefsen, Schmidt's Jahresber., vol. cxcvi, p. 59. 

2 Fleischer, Deutsch. Arch. f. klin. Med., vol. xxix, p. 129. 

3 De la Tourette and Cathelineau, Centralbl. f. d. med. Wiss., 1889, vol. xlviii. 
p. 872. 

4 T. B. Futcher, Jour. Amer. Med. Assoc, 1902, vol. xxxix, p. 1046. 

5 G. Rankin, " Phosphatic Diabetes," Lancet, March 24, 1900. Teissier, These, 
Paris, 1876. 



CHEMISTRY OF THE URINE 385 

Corresponding to the phosphatic retention observed in acute febrile 
diseases an increased elimination is noted during convalescence. 

An increase occurs in the course of cerebrospinal meningitis. 

In a case of pseudoleukemia an increase of 7 grams has been 
noted, while the number of red corpuscles fell from 2,200,000 to 
800,000 in four days. To judge from the very careful observations 
made, there could be no doubt that the high degree of phosphaturia, 
which was limited to the alkaline phosphates, was referable to this 
latter source. In leukemia also very high figures are at times observed. 
Magnus-Levy reports a case in which the patient eliminated about 
15 grams of P 2 5 in fifteen hours. This is exceptional, but other 
observers have noted 5 to 7 grams on repeated occasions. Con- 
sidering the extensive destruction of leukocytes and hence of nucleins 
in leukemia an increased phosphatic excretion appears natural. 

In hemorrhagic purpura Edsall 1 noted a large excretion of P 2 5 : 
6.192 grams. The same observer states that he has seen this also in 
chronic leukemia, as soon as #-ray treatment is begun; at least in 
those cases in which there was the characteristic general response, 
while it did not occur in ,the negative cases. 

While it is apparent that important conclusions cannot be drawn, 
on the whole, from a knowledge of the absolute phosphatic elimina- 
tion, unless it be from a study of the relation existing between the 
excretion of the alkaline and earthy phosphates, a study of the rela- 
tive phos'phatic excretion seems to promise more valuable results. 
According to Ziilzer, 2 a definite amount of the phosphates and of the 
urinary nitrogen is referable to the destruction of albuminous mate- 
rial, so that the relation between the phosphoric acid and the nitro- 
gen must be constant. Another portion, however, is derived from 
lecithin, one of the most important constituents of nerve tissue, 
which contains more phosphorus than the albuminous molecule. 
Whenever, then, the lecithin-containing tissues are more involved 
in the general metabolism than under normal conditions the rela- 
tion will no longer be a stable one. This relation which exists 
between the elimination of nitrogen and phosphoric acid has been 
termed the relative value of phosphoric acid. 

The relative value of phosphoric acid in the urine has been calcu- 
lated as varying from 17 to 20, that of the blood being 3, of muscle 
tissue 12.1, of brain 44, of bone 426 to 430. This value supposes 
the absolute value to vary between 2 and 3 grams pro die. It is found 
according to the following equation: 

N : P 2 5 : : 100 : .r; and x = 100 , • P A 

in which N indicates the amount of nitrogen actually observed, 
P 2 5 the amount of phosphoric acid in the same specimen of urine, 

1 Amer. Jour. Med. Sci., October, 1905. 2 Loc. cit. 

25 



386 THE URINE 

and x the amount of P 2 5 corresponding to 100 grams of N. By 
observing this relative value a much better idea may be formed of 
the metabolic processes taking place in the body in disease than from 
a mere expression of the absolute phosphatic value. 

In acute febrile diseases the relative as well as the absolute dimi- 
nution of the phosphates has been ascribed to a retention, they being 
possibly utilized in the building up of white blood corpuscles. In 
the course of these diseases oscillations in the relative value are fre- 
quently observed ; during convalescence the relative as well as the 
absolute value again rises. 

In accordance with these considerations a diminished relative ex- 
cretion of phosphoric acid should be expected in all cases associated 
with a notable elimination of leukocytes through other channels, as 
in pneumonia, for example, or a storing away of the same, as in 
cases of empyema. The facts observed are in accord with this 
view. 

A relative decrease has further been noted in the various forms of 
anemia, conditions of cerebral excitation, and especially preceding 
an attack of epilepsy. In progressive paralysis following syphilis 
the relative value, at first low, rises greatly after the administration 
of potassium iodide, while the excretion of the earthy phosphates is 
lessened. In chronic cerebral affections, delirium tremens, and acute 
hydrocephalus a relative decrease has been noted. In mania, during 
the period of excitement, both the alkaline and the earthy phosphates 
are found increased, while during the stage of depression, as also in 
melancholia, the alkaline phosphates are diminished and the earthy 
phosphates increased. On the other hand, an increase in the relative 
value has been noted in apoplexy (amounting to 34.3 in one case, 
two days after an attack), brain tumors, tabes, arthritis deformans 
(30), pernicious anemia (23.8 to 58), etc. 1 

Of drugs, bromides appear to diminish the absolute amount of 
phosphoric acid. Cocaine and quinine cause a decrease, and salicylic 
acid an increase. A relative decrease is produced by the cerebral 
excitants, such as strychnine, small doses of alcohol, phosphorus, 
valerian, cold baths, salt-water baths, etc. An opposite effect is 
produced by the cerebral depressants, such as chloroform, morphine, 
chloral, large doses of alcohol, potassium bromide, mineral and 
vegetable acids, prolonged cold baths, Turkish baths, low tempera- 
ture, etc. 

Tests for the Phosphates in the Urine. — The test for the detection 
of the phosphates occurring in the urine depends upon the precipita- 
tion of phosphoric acid by means of ferric chloride as ferric phosphate, 
which is insoluble in cold acetic acid. The same result may be 
accomplished^by^the addition" of a solution of uranyl nitrate; this 

1 Ziilzer u. Edlefsen, loc. cit. 



CHEMISTRY OF THE URINE 387 

gives rise to the formation of uranyl phosphate, which is also in- 
soluble in acetic acid. 

Test. — A few cubic centimeters of urine are acidified with a few 
drops of acetic acid, and treated with a few drops of a solution of 
ferric chloride (1 part of the officinal solution to 10 parts of water), 
when the occurrence of a yellowish- white precipitate will indicate 
the presence of phosphates. 

If a solution containing an acid phosphate of the alkalies is treated 
with an alkaline hydrate, the diacid alkaline phosphate is transformed 
into the monacid salt. This is further changed into the normal salt. 
As the monacid and neutral salts are both readily soluble, the 
solution remains clear. If at the same time, as in the urine, a soluble 
diacid phosphate of the alkaline earths is present, this is likewise 
transformed into the monacid and finally into the neutral salt; the 
latter, however, being insoluble, is thrown down. 

Test for the Earthy Phosphates. — 10 c.c. of urine are 
rendered alkaline with ammonia, when the occurrence of a flocculent 
precipitate will indicate their presence. 

Test for the Alkaline Phosphates. — After having removed 
the earthy phosphates from 10 c.c. of urine, as just described, the 
clear filtrate is acidified with acetic acid and tested with ferric chlo- 
ride or uranyl nitrate, as shown above. 

The alkaline phosphates may also be detected by treating the 
ammoniacal filtrate with a few drops of magnesia mixture (1 part of 
crystallized magnesium sulphate, 2 parts of ammonium chloride, 4 
parts of ammonium hydrate, and 8 parts of distilled water), when 
ammoniomagnesium phosphate, which is almost insoluble in ammo- 
nium hydrate, will be thrown down. 

Quantitative Estimation of the Total Amount of Phosphates . Principle. 
— When a solution of disodium phosphate acidified with acetic acid 
is treated with a solution of uranyl nitrate or uranyl acetate, a dirty- 
looking precipitate of uranyl phosphate is thrown down. It is 
apparent that the quantity of phosphoric acid can be estimated 
accurately, if the solution of uranyl nitrate or acetate is of known 
strength. 

Solutions required: 

1. A solution of uranium nitrate of such strength that 20 c.c. shall 
correspond to 0.1 gram of P 2 5 . 

2. A solution containing sodium acetate and acetic acid. 

3. Tincture of cochineal. 
Preparation of these solutions: 
1. From the equation 

2UO.N0 3 +Na 2 HP0 4 = (UO) 2 HPO, +2NaN0 3 

it is apparent that 2 molecules of uranium nitrate combine with 1 
molecule of disodium phosphate to form uranium phosphate and 



388 THE URINE 

sodium nitrate. The molecular weight of uranium nitrate being 
318 and that of disodium phosphate 142, it is seen that 636 parts 
by weight of the former combine with 142 parts by weight of the 
latter. 

As 20 c.c. of the solution of uranium nitrate shall correspond to 
0.1 gram of P 2 5 , 1000 c.c. must be equivalent to 5 grams of P 2 O s . 
In 142 parts by weight of disodium phosphate there would be present 
71 grams of P 2 O s , equivalent to 636 parts by weight of uranium 
nitrate. The quantity of the latter, then, to be dissolved in 1000 c.c. 
of water will be found from the equation: 636 : 71 : : x : 5; ad x = 
44.78. 

44.78 grams of uranium nitrate are weighed off and dissolved 
in about 900 c.c. of water, the solution being purposely made too 
strong for reasons pointed out in the chapter on Chlorides. In 
order to bring this solution to its proper strength it is necessary to 
titrate with the uranium solution a solution of disodium phosphate 1 
of such strength that each 50 c.c. shall contain 0.1 gram of P 2 5 , 
or 1000 c.c. 2 grams. The molecular weight of Na 2 HP0 4 +12H 2 
being 358, this amount of disodium phosphate in grams is equiv- 
alent to 142 grams of P 2 O s ; the quantity of P 2 5 corresponding to 2 
grams, in terms of Na 2 HP0 4 -f 12H 2 0, is found from the equation: 
358 : 142 : : x : 2; and x = 5.042. This amount of pure, dry, and 
non-deliquescent Na 2 HP0 4 is dissolved in 1000 c.c. of distilled water. 
If non-deliquescent disodium phosphate is not at hand, about 6 or 7 
grams of the salt are dissolved in 1000 c.c. of distilled water; of this 
solution 50 c.c. are evaporated in a weighed platinum dish, and the 
residue gently heated, the disodium phosphate being thereby trans- 
formed into sodium pyrophosphate, Na 4 P 2 O r The molecular weight 
of Na 4 P 2 7 being 266, this corresponds to 142 grams of P 2 5 . If 
the solution is of the correct strength — 1 e., containing 0.1 gram of 
P 2 5 in 50 c.c. of water — the residue should weigh 0.1873 gram, as 
is seen from the equation : 142 : 266 : : 0.1 : x; and x = 0.1873. Sup- 
posing, however, that the residue weighs 0.1921 gram, it is manifest 
that the solution is too strong, and must be diluted, the degree of 
dilution being ascertained according to the equation: 0.1873 : 1000 
: : 0.1921 : x; and # = 1025; i. e., 1000 c.c. of the solution must be 
diluted to 1025 c.c. to make it of the proper strength. 

In the case given, 50 c.c. were used; the 950 c.c. are then diluted 
with the amount of water found from the equation: 1000 : 1025 : : 
950 : x; and #=973.75. Having thus obtained a solution of diso- 
dium phosphate of such strength that each 50 c.c. shall contain 0.1 
gram of P 2 5 , this is titrated with the uranium solution, which 
has been made too strong, in order to determine the amount of 

1 A solution of chemically pure crystallized monopotassium phosphate can also 
be used for standardization (Sutton's Volumetric Analysis, 8th ed., p. 316), 



CHEMISTRY OF THE URINE 389 

water that must be added to the latter. To this end, a burette is 

filled with the uranium solution; 50 c.c. of the disodium phosphate 

solution are treated with a few drops of the tincture of cochineal 

and 5 c.c. of the acetic acid mixture (see below). This mixture is 

heated in a beaker, and as soon as the boiling point has been reached 

titrated with the uranium solution until a trace of a greenish color 

is noticed in the precipitate which does not disappear on stirring. 

This point having been accurately determined by means of a second 

titration, the number of cubic centimeters of distilled water with 

which the remaining solution must be diluted is determined accord- 

N. d 
ing to the formula: C= — : — , in which C represents the number 

of cubic centimeters which must be added, N the number of cubic 
centimeters remaining after the test titration, n the number of cubic 
centimeters consumed in one titration to bring about the end reac- 
tion, and d the difference between the number of cubic centimeters 
used in one titration and that theoretically required. 

The amount of distilled water necessary for dilution is now added 
and the solution again .tested, when 20 c.c. will correspond to 0.1 
gram of P 2 5 . 

2. The acetic acid mixture is prepared by dissolving 100 grams 
of sodium acetate in a little Water, adding 30 grams of glacial acetic 
acid and diluting the whole to 1000 c.c. 

3. Tincture of cochineal. This may be prepared as follows: A 
few grams of cochineal granules are digested at ordinary tempera- 
tures with 250 c.c. of a mixture of 3 volumes of water and 1 volume 
of 94 per cent, alcohol. The solution is then decanted and ready 
for use. The residue may be utilized in the preparation of a fresh 
supply of the tincture. 

Application to the Urine. — 50 c.c. of clear filtered urine are 
treated with 5 c.c. of the acetic acid mixture, the object being to 
transform any monacid sodium phosphate present into diacid sodium 
phosphate, and to neutralize any nitric acid that may be formed 
during the titration, as otherwise the nitric acid would cause a partial 
solution of the precipitated uranyl phosphate. A few drops of the 
tincture of cochineal are added, when the mixture is heated to the 
boiling point and titrated as described above. Two titrations are 
usually required. 

Should it be desired to use potassium ferrocyanide as an indicator, 
the uranium solution must have been standardized with the same 
indicator, as errors will otherwise arise. The technique is simple. 
A number of droplets of the potassium ferrocyanide solution (about 
5 per cent.) are placed on a piece of white filter paper. After every 
addition of the uranium solution to the boiling urine a droplet of the 
mixture is placed upon the ferrocyanide stain. The end reaction is 
indicated by the occurrence of a brown color. 



390 THE URINE 

The results are calculated as follows: Supposing 15 c.c. of the 
uranium solution to have been used, the corresponding amount of 
P 2 5 in 50 c.c. of urine is found from the equation: 20: 0.1 : : 15: x; 
and x = 0.075. The percentage amount would, hence, be 0.075 X 
2 = 0.15. Supposing the total amount of urine to have been 2000 
c.c, the elimination of P 2 5 would correspond to 3 grams. 

The presence of sugar and albumin does not interfere with the 
method. 

Separate Estimation of the Earthy and Alkaline Phosphates. — 
If the alkaline and earthy phosphates are to be determined separately, 
the total amount of P 2 5 is estimated in one portion of the urine, 
while the P 2 5 in combination with the alkaline earths is determined in 
another, as follows: 200 c.c. of filtered urine are made strongly alkaline 
with ammonium hydrate and set aside, covered, for several hours, 
when the earthy phosphates thus preciptated are collected on a filter, 
washed with dilute ammonia (1 to 3), and then transferred to a beaker, 
with the aid of a little water containing a few drops of acetic acid, 
by perforating the filter. They are then dissolved with as little 
acetic acid as possible, diluted to 50 c.c. with distilled water, and 
titrated with the uranium solution as described. The difference 
between the total amount of P 2 O s and the amount thus obtained 
indicates the quantity of alkaline phosphates present. 

Removal of the Phosphates from the Urine. — Whenever it is 
necessary to remove the phosphates from the urine in the course of an 
analysis, as is frequently the case, the urine is rendered alkaline by the 
addition of the hydrate of an alkaline earth and precipitated with a 
soluble calcium or barium salt. They may also be precipitated by 
means of neutral or basic lead acetate, in which case the excess of lead 
is removed by means of hydrogen sulphide or dilute sulphuric acid. 



The Sulphates. 

The sulphuric acid found in the urine is derived essentially from 
the albuminous material which is constantly broken down in the 
body, a very small portion only of the inorganic sulphates being refer- 
able to the mineral constituents of the food. As was pointed out in the 
chapter on Reaction, sulphuric acid is constantly produced in the 
body, and, coming into contact with the so-called neutral phosphates 
present in almost all the tissues, transforms these into acid phos- 
phates, both appearing in the urine. The alkaline carbonates, which 
are derived from the organic salts ingested by a process of oxidation, 
are also attacked by the sulphuric acid. 

As the amount of food ingested is gradually diminished a point is 
reached when the body most tenaciously holds any alkaline salts that 
may still be present. A new source for the neutralization of the 



CHEMISTRY OF THE URINE 391 

acid is then found in the ammonia, which would otherwise have 
been eliminated as urea. 

While the greater portion of the sulphuric acid excreted in the 
urine is found in the form of mineral sulphates, about one-tenth of 
the total amount may be shown to be in combination with aromatic 
substances belonging to the oxy-group; most important among these 
are the salts of phenol, indoxyl, and skatoxyl. 

Indoxyl and skatoxyl, as will be shown later, are derived from 
indol and skatol, which, together with phenol, are formed during 
the process of intestinal putrefaction. Their amount increases and 
decreases with the degree of putrefaction, and hence serves as an 
index of its intensity. 

The mineral sulphates have been termed preformed sulphates in 
contradistinction to the others, which are known as conjugate or 
ethereal sulphates. In the following pages the former will be desig- 
nated by the letter A, the conjugate sulphates by the letter B, and 
the total sulphates as A+B. 

The amount of A-{-B excreted in the twenty-four hours by a 
normal individual varies between 2 and 3 grams, the ratio of A to 
B being as 10 to I. 1 

From what has been said, it is apparent that the elimination of 
sulphates is largely dependent upon the degree of albuminous destruc- 
tion taking place in the tissues and fluids of the body, and hence 
to a certain extent upon the quantity of proteid material ingested, 
the mineral sulphates occurring in such small amount in the food as 
scarcely to affect the quantity excreted. Secondarily; the degree of 
intestinal putrefaction plays a role. During starvation A-\-B is 
diminished, this diminution affecting A especially; in some cases B 
may be considerably increased. 2 

An increase in the elimination of the total sulphates is observed, 
as would be anticipated, in all cases in which an increased tissue 
destruction is taking place, as in acute febrile diseases. It must 
be remembered, however, that the quantity excreted is then not 
always greater than during convalescence, the diet remaining the 
same. Here, as elsewhere, in urinary studies, it is necessary to dis- 
tinguish between a relative increase and an absolute decrease. In 
pneumonia and acute myelitis the highest figures have been observed, 
the increased elimination during the febrile period being especially 
marked : 3 

Fever diet. Full diet. 

Fever. No fever. No fever. 

Pneumonia 3.51 gm. 1.47 gm. 2.25 gm. 

Acute myelitis . . . . 2.62 gm. 1.52 gm. 2.33 gm. 

1 v. d. Velden, Virchow's Archiv, vol. vii, p. 343. 

2 Clare, Inaug. Diss., Dorpat, 1854. 

3 P. Furbringer, Virchow's Archiv, vol. lxxiii, p. 39. 



392 THE UEINE 

During convalescence the excretion of the sulphates is diminished, 
a retention analogous to that of the chlorides and phosphates taking- 
place. In contradistinction to the latter salts, it is in all probability 
not the mineral matter proper that is demanded by the body, but the 
sulphur-containing albuminous material. 

A considerable elimination of A^B has also been observed in 
leukemia, in which an average of 2.46 grams is excreted, as com- 
pared with 1.51 grams by a healthy individual receiving the same 
amount and kind of food. In one case of acute leukemia 5.8 grams 
were eliminated on the day preceding death. 1 

In diabetes mellitus, diabetes insipidus, esophageal carcinoma, 
progressive muscular atrophy, pseudohypertrophic paralysis, and 
eczema an increased elimination has likewise been observed, while 
in chronic renal diseases a diminished excretion is the rule. 

A study of the elimination of the conjugate sulphates and of the 
relation existing between A and B in disease is still more important 
than that of the total sulphates; but in both cases the data available 
are scanty, and further observations are urgently needed, v. Noor- 
den regards the elimination of more than 0.3 gram of conjugate 
sulphates in the twenty-four hours as excessive, the patient being 
on an ordinary mixed diet. 

The conjugate sulphates, as would be expected, are increased in 
all cases of increased intestinal putrefaction. 2 In coprostasis the 
result of carcinoma the ratio of the preformed to the conjugate sul- 
phates, normally 10, may dimmish enormously. In one case, reported 
by Kast and Baas, 3 it fell to 2, but rose to 7 and 8, and finally to 9.5 
and 15 after an artificial anus had been established. I have observed 
a drop to 1.5 in a case of volvulus of ten days' standing. H. Baldwin 
notes a case of pernicious vomiting of pregnancy in which the factor 
A : B was 1.9; following abortion it fell to 4 and a little later to 
5.4. Biernacki 4 found an increase in the elimination of conjugate 
sulphates amounting to from 0.15 to 0.5 gram pro die in cases of 
chronic parenchymatous nephritis, going hand in hand apparently 
with a decrease in the secretion of hydrochloric acid by the stomach; 
the normal amount, according to his observations, varies from 0.1973 
to 0.2227 gram. In one case B fell from 0.4382 to 0.1505 during 
the administration of hydrochloric acid, to increase again to 0.4127 
upon its discontinuance. 

In accord with these observations are those of Wasbutzki and 

1 Ebstein, Deutsch. Arch. f. klin. Med., vol. xliv, p. 346. 

2 Blumenthal has called attention to the fact that this is not necessarily the 
case, and that an acid fermentation may occur in lieu of the formation of aromatic 
products. He hence suggests that at times it may be necessary to estimate the 
volatile fatty acids also. 

3 Munch, med. Woch., 1888. 

4 Deutsch. Arch. f. klin. Med., vol. lxix. 



CHEMISTRY OF THE URINE 393 

Kast. 1 The former found an increased elimination of B in cases of 
intense bacterial fermentation taking place in the stomach, while 
hydrochloric acid was either totally absent or present in greatly 
diminished amount. A diminished elimination was observed in cases 
of intense torular fermentation, rryperchlorhydria existing at the 
same time. In the absence of hydrochloric acid a normal or even 
a slightly diminished amount was observed in cases of intense acid 
fermentation, lactic acid and butyric acid being present in large 
quantities. 

By neutralizing the gastric juice with large doses of sodium 
bicarbonate Kast was able to bring about a marked increase in the 
elimination of B, the ratio A : B having fallen from 10.3 to 16.1 to 
2.9 to 6.1. Personal observations have led me to the same con- 
clusion. 2 (See also chapter on the Aromatic Bodies.) 

In obstructive jaundice the excretion of B is likewise increased; 
it returns to the normal as soon as the permeability of the biliary 
passages has again become established. The total sulphates were 
found diminished in cases of non-obstructive jaundice. 3 In 
B ohm's 4 cases of catarrhal jaundice the excretion of conjugate 
sulphates varied between 0.4 and 0.7 gram. Of interest in this con- 
nection are the observations of Muller, 5 who notes the elimination 
of 0.29, 0.24, and 0.28 gram of conjugate sulphates on three con- 
secutive days in a case of total obstruction of the biliary duct in 
consequence of a stone. The patient during this period was on a milk 
diet, and there can be little doubt that the low values are here refer- 
able to the pure lactic acid producing organisms crowding out the 
colon bacilli. On a meat diet the same patient passed 0.48 and 
0.51 gram. Other observers have obtained less constant results in 
their cases of catarrhal jaundice. In cases of hepatic cirrhosis and 
malignant disease of the liver Eiger^ and Hop adze 7 found increased 
amounts of conjugate sulphates. 

In cases of diarrhea A-\-B, as well as B, is diminished, while A:B 
is increased. 

Of drugs, large doses of morphine, potassium bromide, sodium 
salicylate, and antifebrin appear to cause an increased elimination of 
the total sulphates, while alcohol slightly diminishes the excretion. 

Most important are the observations which have established a 
diminished excretion of the conjugate sulphates following ingestion 
of the terpenes and camphor, Karlsbad and Marienbad water, which 

1 Kast, Festsch. z. Eroffnung d. neuen allgem. Krankenhauses, Hamburg, 
1889. Wasbutzki, Arch. f. exper. Path. u. Pharmakol., vol. xxvi. 

2 C. E. Simon, Amer. Jour. Med. Sci., 1895, vol. ex. 

3 Ziilzer, Unters. uber d. Semiol. d. Harns, Berlin, 1884. 

4 Deutsch. Arch. f. klin. Med., 1901, vol. lxxi, p. 73. 

5 Zeit. f. klin. Med., 1887, vol. xii. 

6 Inaug. Diss., St. Petersburg, 1893. 

7 Wratsch, 1893, Nos. 48 to 50. 



394 THE URINE 

latter two, however, at first cause an increase. Kefir, in doses of 
from 1 to 1.5 liters pro die, has proved a most excellent remedy with 
which to combat this type of intestinal putrefaction. Injections of 
tannic acid and of a saturated solution of boric acid apparently produce 
little effect unless the dose is so large as to cause symptoms of 
poisoning. 

Tests for the Sulphates in the Urine. — The detection of the mineral 
and the conjugate sulphates in the urine depends upon the fact that 
the sulphates of the alkalies are precipitated by barium chloride as 
insoluble barium sulphate. In the urine the addition of barium 
chloride at the same time causes a precipitation of the phosphates. 
These must be kept in solution by the addition of an acid, acetic 
acid being employed for this purpose whenever the presence of the 
mineral sulphates is to be demonstrated; hydrochloric acid is inad- 
missible, as it would cause the decomposition of the conjugate sul- 
phates and set free the sulphuric acid thus held. 

To test for the mineral sulphates, a few cubic centimeters of urine 
strongly acidified with acetic acid are treated with a few drops of a 
solution of barium chloride, when in their presence a cloud or a 
white precipitate of barium sulphate will occur. 

To test for the conjugate sulphates, 25 c.c. of urine are treated 
with about the same volume of an alkaline barium chloride mixture 
(2 volumes of a solution of barium hydrate and 1 volume of a solu- 
tion of barium chloride, both saturated at ordinary temperatures) 
and filtered for a few minutes, the preformed sulphates as well as 
the phosphates being thus removed. The filtrate is then strongly 
acidified with hydrochloric acid and boiled; the occurrence of a pre- 
cipitate is referable to conjugate sulphates. 

Quantitative Estimation of the Sulphates. — The principle of the 
method is the same as that just described, the mineral sulphates 
forming an insoluble precipitate of barium sulphate directly when 
treated with barium chloride, while the conjugate sulphates do so 
only upon decomposition with strong hydrochloric acid under the 
application of heat. In order to estimate the mineral and conjugate 
sulphates, it is best to determine the total sulphates in one portion 
and the conjugate sulphates in another, the difference between the 
two giving the mineral sulphates. 

Quantitative Estimation of the Total Sulphates (Folin). — 50 c.c. of 
clear, filtered urine are treated with 5 c.c. of concentrated hydro- 
chloric aid and 5 c.c. of a 4 per cent, solution of potassium chlorate. 
The mixture is boiled until it is colorless (five to ten minutes) and 
then treated, while still boiling, with 25 c.c. of a 10 per cent, solution 
of barium chloride, drop by drop. It is kept on a hot-water bath or 
on an asbestos plate hot (but not boiling) for one-half to one hour. 
The precipitate is now collected on a Schleicher and Schiill filter, 
the weight of the ash of which is known (No. 589). Care should be 



CHEMISTRY OF THE URINE 395 

taken. never to allow the filter to run dry, and small amounts of hot 
water must be added to the last cubic centimeters remaining, the final 
traces being placed upon the filter with the aid of a rubber-tipped 
glass rod. The precipitate is washed with hot water for a half-hour, 
and at intervals of a few minutes hot ammonium chloride solution 
(5 per cent.) is substituted for the water, so that in all five or six 
additions of ammonium chloride take place in the course of the first 
twenty minutes' washing. In the end a specimen of the washings 
must no longer be rendered cloudy, even on standing a few minutes, 
upon adding a drop of dilute sulphuric acid. 

The paper filter is partially dried by folding and pressing gently 
between filter paper. It is then placed in a weighed crucible, 
covered with 3 to 4 c.c. of alcohol, and the alcohol ignited. The ash 
is heated, at first moderately, and almost completely covered with the 
lid, then only half covered, for five to seven minutes, until the contents 
of the crucible are white. The crucible, when cooled, is placed in 
a desiccator and weighed, the difference between the first and the 
second weighing giving the weight of the barium sulphate obtained 
from 50 c.c. of urine. 

Quantitative Estimation of the Conjugate Sulphates (Folin). — 200 
c.c. of urine (diluted to a liter if necessary) are treated with 100 c.c. 
of a 10 per cent, solution of barium chloride, at ordinary tempera- 
ture. The mixture is set aside for twenty-four hours and the clear 
supernatant fluid poured into a dry beaker by decanting. This pre- 
liminary decantation is necessary, as the barium sulphate precipitate 
will otherwise go through the paper. The decanted liquid is filtered, 
150 c.c. of the clear filtrate, representing 100 c.c. of urine, measured 
into an Erlenmeyer flask, treated with 10 to 15 c.c. of concentrated 
hydrochloric acid and 10 to 15 c.c. of a 4 per cent, solution of potas- 
sium chlorate. The mixture is then heated to boiling and kept upon 
a boiling water bath until the barium sulphate has settled and the 
supernatant fluid is clear. The precipitate is filtered off, washed, 
dried, and weighed, as described above. The weight thus obtained, 
deducted from the amount found according to the first method, indi- 
cates the amount referable to the mineral sulphates. The molecular 
weight of BaS0 4 being 232.82, that of SO s 79.86, of H 2 S0 4 97.82, 
and of S 32, the figure expressing the amount of H 2 S0 4 , S0 3 , or S, 
corresponding to 1 gram of BaS0 4 , is found according to the follow- 
ing equations: 

232.82 : 79.86 : : 1 : x; and x = 0.34301. /. 1 gram of BaS0 4 = 
0.34301 gram of S0 3 . 

232.82 : 97.82 : : 1 : x; and x = 0.42015. .\ 1 gram of BaS0 4 = 
0.42015 gram of H 2 S0 4 . 

232.82 : 32 : : 1 : x; and x =0.13744. .'. 1 gram of BaS0 4 = 
0.13744 gram of S. 

To calculate results, it is only necessary to multiply the weight of 



396 THE URINE 

the BaS0 4 by 0.34301, 0.42015, or 0.13744, in order to ascertain the 
amount of sulphuric acid contained in 50 c.c. of urine, in terms of 
S0 3 , H 2 S0 4 , or S, respectively. 

Literature. — E. Salkowski, Zeit. f. physiol. Chem., 1886, vol. x, p. 346; and 
Virchow's Arch., 1888, vol. lxxix, p. 551. O. Folin, Amer. Jour. of. Physiology, 
1902, vol. vii, p. 152. 

Neutral Sulphur. 

While the greater portion of the sulphur of the body is eliminated 
in an oxidized form, small amounts of non-oxidized sulphur bodies 
are likewise found in every urine. They are collectively spoken of 
as the neutral sulphur of the urine, and under normal conditions 
constitute from 12 to 15 per cent, of the total sulphur. The rela- 
tion existing between the oxidized and the neutral form is, however, 
inconstant, and varies with the character of the diet, the degree of 
the proteid metabolism, etc. 

Of the nature of the neutral sulphur bodies which occur in nor- 
mal urine, comparatively little is known. At the present time we 
are acquainted with only two substances belonging to this order, 
viz., certain sulphocyanides and cystein, or a body which is closely 
related to it. The greater portion of the sulphocyanides is undoubt- 
edly derived from the saliva that has been swallowed and absorbed, 
while a smaller amount may be referable to the trace which is said 
to be present in normal, uncontaminated gastric juice. The amount 
of sulphur which is present in this form represents about one-third 
of the total quantity of the neutral sulphur. Cystein probably 
is an intermediary product of the normal metabolism of proteid 
material. Under normal conditions, however, the greater portion 
is oxidized to sulphuric acid, and traces only escape to be eliminated 
as such. 

Whether or not taurocarbaminic acid, which is a derivative of 
taurin, is a constant constituent of the urine remains an open question, 
but is very probable. We know, as a matter of fact, that the amount 
of neutral sulphur undergoes a distinct diminution in animals when 
the bile is prevented from entering the intestinal canal by establish- 
ing an external fistula. Under pathological conditions a correspond- 
ing increase is observed in cases of biliary obstruction, and the 
amount of neutral sulphur may then reach 40 per cent, of the total 
sulphur. 

Thiosulphates, which are normally present in the urine of dogs 
and cats, do not occur in human urine under normal conditions. 
That they may be present in disease has been shown by Strumpell, 
who found them in a case of typhoid fever. Further observations, 
however, are wanting. 

Another sulphur body belonging to this class, which Abel dis- 



CHEMISTRY OF THE URINE 397 

covered in the urine of dogs, and which appears to be identical with 
ethyl sulphide, has not been found in the urine of man. 

The greatest increase in the amount of the neutral sulphur is 
observed under certain conditions associated with the appearance 
of cystin. Normally this is not present in the urine, while traces 
of cy stein, or a closely related substance, as I have already stated, 
are found. Cystin is of albuminous origin, and as a matter of fact 
it has been ascertained that all of the loosely combined sulphur 
and even a portion of the firmly combined form exists in the albu- 
mins in the form of the cystin complex. According to Baumann 
and v. Udranszky, its appearance in the urine is closely connected 
with the formation of certain diamins, viz., cadaverin> putrescin, 
and a third diamin which is probably identical with saprin or neu- 
ridin. As these diamins were hitherto supposed to result only from 
the action of certain specific bacteria upon albuminous material, 
cystinuria was regarded as evidence of a definite infectious pro- 
cess. It is to be noted, however, that cystin itself does not occur 
in the feces, and that diaminuria does not necessarily accompany 
the cystinuria. As the result of personal observations I have been 
led to the conclusion that a causal connection does not exist between 
the two conditions, and that the diamins in question can be pro- 
duced in the body tissues directly without the intervention of micro- 
organisms. I regard cystinuria essentially as a metabolic anomaly, 
the result of a specific insufficiency on the part of certain tissues 
(liver) of the body. The condition may be temporary, but as a rule 
it is permanent. It may occur among several members of the same 
family, but it is noteworthy that no case has been reported in 
which a parent and child were cystinuria Consanguinity among 
parents, which is not infrequently observed in*eases of alkaptonuria, 
is the exception in cystinuria. 

In this connection it is interesting to note that according to Lowy 
and Neuberg 1 the cystinuric is not able to oxidize other mono- and 
diamino acids when given by the mouth, and that tyrosin and aspar- 
tic acid will reappear as such, while ly sin and arginin are eliminated 
as cadaverin and putrescin. Folin and I have not been able to verify 
this observation so far as tyrosin goes. Abderhalden 2 , on the other 
hand, found tyrosin and leucin in the urine of a cystinuric, who had 
not been fed any tyrosin as such. 

The amount of neutral sulphur which may be met with in cystin- 
uria is subject to wide variation, but not infrequently exceeds 30 
per cent, of the total sulphur. As a general rule, the amount of 
cystin eliminated in the twenty-four hours is less than 0.5 gram. 
At times, however, larger quantites are found, and on one occasion 

1 Zeit. f. physiol. Chem., vol. xliii, p. 338. 

2 Abderhalden and Schittenhelm, ibid., vol. xlv, p. 468. C. E. Simon, 
ibid., vol. xlv, p. 24, 



398 THE URINE 

I obtained more than 1 gram. Clinically it is of interst in so far as 
its continued production may give rise to the formation of calculi. 

Unless cystin occurs as a deposit, its presence will scarcely be 
suspected. The substance, however, may occur also in solution, 
and it not infrequently happens that attention is first drawn toward 
its existence in this state owing to the marked odor of hydrogen 
sulphide which such urines develop on standing (see Hydrothion- 
uria). If acetic acid is then added in excess, the characteristic 
hexagonal plates may crystallize out. The same result is obtained 
by allowing the urine to undergo aminoniacal decomposition, as 
cystin is insoluble in solutions of ammonium carbonate. 

Structurally cystin is the disulphide of cystein, which latter is 
«-amino-/?-thiolactic acid. On reduction it is transformed into 
cystein according to the equation: 

CH 2 S S.CH 2 CH 2 .SH 

I I I 

CH.NH, CH.NH 2 + 2H = 2CH.NH 2 

I I I 

COOH COOH COOH 

Cystin crystallizes in hexagonal plates which are quite characteristic, 
and not likely to be confounded with other crystalline elements that 
may be present in urinary sediments. If doubt should arise, their 
solubility in ammonia and hydrochloric acid, and their insolubility 
in acetic acid, water, alcohol, and ether, will lead to their identifi- 
cation. 

The quantitative estimation of cystin is rather unsatisfactory, as 
no method is known which yields reliable results. On the whole, it 
is perhaps best to determine the neutral sulphur, and to refer the 
increase beyond its normal value to the presence of cystin. 

Quantitative Estimation of the Neutral Sulphur in the Urine. — In one 
portion of urine the oxidized sulphur, viz., the mineral and the con- 
jugate sulphates, are estimated as described. In a second portion 
the total sulphur is determined, the difference indicating the amount 
of the neutral sulphur. 

To determine the total amount of sulphur the following method is 
most conveniently employed: 

Method of Hbhnel-Glaser (modified by Modrakowsky 1 ) : 1 or 2 
grams of sodium peroxide are placed in a nickel dish, and covered 
with 50 c.c. of urine, added drop by drop. The fluid is evaporated 
to a syrup on a water bath, and further treated with 2 to 3 grams of 
the peroxide, which is added slowly while stirring. As soon as the 
reaction, which at first is quite vigorous, has subsided somewhat, the 
dish is removed from the water bath and heated with a small alcohol 
lamp. If necessary, 1 to 3 grams more of the peroxide are added. 

1 Zeit. f. phys. Chem., 1903, vol. xxxviii, p. 562. 



CHEMISTRY OF THE URINE 399 

The mass now forms brown drops and finally becomes thick; this 
ends the reaction. On cooling, the fusion is dissolved in hot water; 
the solution is filtered and feebly acidified with hydrochloric acid. 
Barium chloride is then added and the process continued as above 
described (Estimation of Sulphates). 

Literature. — E. Salkowski,Virchow's Archiv,vol. lxvi, p. 313, and vol. cxxxvii, 
p. 381. Goldmann u. Baumann, " Zur Kenntniss der Schwefelhaltigen Ver- 
bindungen des Harns," Zeit. f. physiol. Chem., vol. xii, p. 254. E. Salkowski, 
Virchow's Archiv, vol. lviii, p. 461. " J. Munk, ibid., vol. lxix, p. 354; and Deutsch. 
med. Woch., 1877, No. 46. O. Schmiedeberg, "Ueber das Vorkommen von 
Unterschwefliger Saure im Harn," Arch. d. Heilk, vol. viii, p. 425. A. Strumpell, 
ibid., vol. xvii, p. 390. J. Abel, "Ueber das Vorkommen von Ethylsulfid im 
Hundeharn," etc., Zeit. f. physiol. Chem., vol. xx, p. 253. (See also Cystinuria 
and Hydrothionuria.) C. E. Simon, " Cystinuria and its Relation to Diaminuria," 
Amer. Jour. Med. Sci., 1900, vol. cxix, p. 39. C. E. Simon and M. W. Lewis, 
" Transitory Cystinuria," ibid., 1902, vol. cxxiii, p. 838. C. E. Simon and D. G. 
J. Campbell, "A Contribution to the Study of Cystinuria," Johns Hopkins Hospital 
Bull., 1904, vol. xv, p. 365. 



Urea. 

Urea is the most important nitrogenous constituent of the urine, 
and normally represents from 85 to 86 per cent, of the total amount 
of nitrogen which is eliminated by the kidneys. Chemically, it may 
be regarded as carbamide — i. e., as the amide of carbonic acid— and 
is represented by the formula 

/NH 2 

\nh 2 . 

It is thus a comparatively simple substance, and the question natu- 
rally arises : What relation does urea bear to the complex albuminous 
molecule from which it is derived? According to the older concept 
of the process of albuminous digestion this leads to the formation of 
albumoses and peptones, which latter were regarded as a unity and 
as very complex substances. From these bodies the reconstruction 
of the albuminous molecule then supposedly took place in the intesti- 
nal mucous membrane, whence the resulting albumin found its way 
into the blood and lymph. Here it existed as so-called circulating 
albumin, in contradistinction to the organized albumin of the cells. 
Voit further taught that the circulating albumin is broken down in 
the tissues at large, through the special activity of the living proto- 
plasm, but without becoming an integral part of the cells before its 
destruction. Pfliiger, on the other hand, took the contrary view 
according to which the circulating albumin must become part and 
parcel of the cells before it can undergo katabolic disintegration. In 
either event it was generally accepted that urea was derived from the 
tissues of the body at large and to a great extent from the muscles. 
Regarding the nature of its intermediary antecedents, Drechsel sup- 



400 THE URINE 

posed that amino-acids first result by hydrolysis in the tissues, and 
that ammonia, water, and carbon dioxide are then formed from these 
by oxidation. Ammonia and carbon dioxide then combine and form 
ammonium carbamate, which is carried to the liver and is there trans- 
formed into urea through loss of water. These stops are represented 
by the equations: 

1. CH 2 .NH 2 .COOH + 30 = NH 3 +2C0 2 + HX>. 

Glycocoll. 

/NH 2 

2. C0 9 +2NH 3 = CO( 

\O.NH 4 . 
/NH 2 

3. CO( /NH 2 

\O.NH 4 — H 9 = C0( 

\NH 2 . 
Urea. 

As a matter of fact it is well known that the ingestion of amino- 
acids leads to an increased elimination of urea and that the liver plays 
an important role in its final formation. But there is still much 
doubt whether amino-acids are formed in the tissues at large to such 
an extent as the older theories of Voit and Pfliiger would demand. 
It is rather significant that normally they are scarcely ever encoun- 
tered in the tissues of the mammalian organism, and for some years 
past there has been a growing tendency to regard ammonium para- 
lactate as the principal form in which the greater portion of the nitrogen 
leaves the tissues. In the liver this is then supposedly transformed 
into ammonium carbonate, from which the urea results with the inter- 
mediary formation of ammonium carbamate. 

This hypothesis has certain facts in its favor. We thus find that 
after extirpation of the liver in geese the uric acid, which in birds plays 
the same part as the urea in mammals, disappears and is largely 
replaced by ammonium lactate. In diseases of the liver, moreover, 
in which an extensive destruction of the parenchyma is taking place, 
as in some cases of acute yellow atrophy, in phosphorus poisoning, 
etc., the elimination of urea is diminished, and in its place a cor- 
responding amount of ammonia in combination with lactic acid is 
found. In dogs in which the liver has been in part excluded from 
the general circulation by the establishment of an Eck fistula, and 
in which the hepatic artery has at the same time been ligated, the 
elimination of urea is much diminished, while that of ammonia 
increases rapidly so soon as the first symptoms of illness appear in 
the animals. From these observations it is apparent also that the 
synthesis of urea takes place in the liver. This is further proved 
by the fact that on transfusion of isolated livers of dogs with blood 
to which ammonium carbonate or ammonium lactate has been added, 
urea is formed as a result. In other organs of the body this synthesis 
apparently does not occur, but there is evidence to show that at least 



CHEMISTRY OF THE URISE 401 

a small amount of urea originates elsewhere within the body through 
processes of hydrolysis. This amount, however, is unquestionably 
slight. That a fraction, moreover, is formed from uric acid, and in 
the last instance from the xanthin bases through processes of oxida- 
tion, can scarcely be doubted, but this transformation apparently also 
takes place in the liver. 1 

Of late Folin 2 has formulated a theory of proteid metabolism which 
in my judgment is more in conformity with our present knowledge of 
proteid digestion and more satisfactorily explains many questions 
connected with the subject of nitrogenous metabolism than any 
other. He distinguishes sharply between tissue metabolism or en- 
dogenous metabolism which tends to be constant, and exogenous or 
intermediate metabolism which is variable. As essential nitrogenous 
end product in the first instance he regards kreatinin, the elimination 
of which he finds practically constant for one and the same individual. 
Urea, according to his conception, is the principal nitrogenous end 
product in the case of the exogenous metabolism. According to his 
idea the amino-acids which result on gastro-intestinal digestion, in 
so far as they are not needed immediately to make up for tissue loss 
in nitrogen, are at once desamidized in the liver. The non-nitrog- 
enous remainder is then utilized in the formation of fats and carbo- 
hydrates, while the amino group gives rise to the formation of urea. 

In this manner the presence of the large amounts of ammonium com- 
pounds which are found in the portal blood during digestion is well 
explained. But as Howell remarks, while a portion and perhaps a 
large portion of the urea arises from this early hydrolysis of the pro- 
teids of the food we must admit also that ammonium compounds may 
be formed in the tissues of the body generally, probably by a similar 
process of hydrolysis followed by oxidation. This would suggest 
itself especially under pathological conditions where the amount of 
urea nitrogen may be in excess of that corresponding to the ingested 
food. 

It has been stated that 84 to 86.6 per cent, of all the nitrogen 
eliminated in the urine is in the form of urea, the remaining 13.4 
per cent, being excreted as uric acid, hippuric acid, kreatinin, xanthin 

1 The origin of urea: O. Schultzen u. M. Nencki, Zeit. f. Biol., 1872, vol. viii, 
p. 124. E. Salkowski, Zeit, f. physiol. Chem., 1879, vol. iv, p. 100. v. Knieriem, 
Zeit. f. Biol., 1874, vol. x, p. 279. E. Salkowski, Zeit, f. physiol. Chem., 1877, 
vol i, p. 38. Hoppe -Sevier, Physiol. Chem., 1881, p. 810. " Drechsel, Jour. f. 
prakt. Chem., vol xv, p. 417: vol. xvi, pp. 169 and 180, and vol. xxii, p. 476. 
M. Hahn, V. Massen, M. Nencki, and J. Pawlow, " La fistula d'Eck," etc., Arch, 
d. Sci biol. de St. Petersburg, 1892, vol. i. 

Seat of formation: W. v. Schroder, Arch. f. exper. Path. u. Pharmakol., 1882. 
vol. xv, p. 364. W. Salomon, Virchow's Archiv, 1884, vol. xcvii, p. 149. Min- 
kowski, " Ueber d. Einfluss d. Leberextirpation auf d. Stoffwechsel," Arch. f. 
exper. Path. u. Pharmakol., 1886, vol. xxi, p. 41, and 1893, vol. xxxi, p. 214. 

2 C. Voit, Phvsiol. d. allg. Stoffwechsels u. d. Ernahrung. Hermans' Hand- 
buch d. Phvsiol., 1881, vol. vi, I, p. 301. O. Folin, Amer. Jour, of Physiol., 
1905, vol. xiii. 

26 



402 THE URINE 

bases, etc. It might hence be supposed that an accurate idea of 
the degree of tissue destruction could be formed from a quantita- 
tive estimation of urea. This, however, is not the case, and especially 
in pathological conditions, as the quantitative relations existing 
between the excretion of urea and the remaining nitrogenous con- 
stituents are subject to wide variation. In acute yellow atrophy, 
for example, urea may disappear entirely from the urine, the nitrogen 
being eliminated in the form of other compounds (leucin, tyrosin, 
glycocoll, etc.). Whenever it becomes desirable, then, to gain an 
accurate insight into the degree of proteid destruction or proteid 
assimilation — in other words, into the nitrogenous metabolism — taking 
place in the body, it is necessary to resort to a quantitative determina- 
tion of the total amount of nitrogen excreted by the kidneys; the 
quantity found is then conveniently expressed in terms of urea. At 
the same time it is customary to express the amount of proteid tissue 
which is destroyed, as muscle tissue, as this serves as a fair type of 
body tissue in general. 

As 100 grams of lean muscle tissue contain about 3.4 grams of 
nitrogen, corresponding to 7.286 grams of urea, 1 gram of the latter 
is equivalent to 13.72 grams of muscle tissue. It is, hence, only 
necessary to multiply the quantity of urea eliminated in the twenty- 
four hours, corresponding to the total amount of nitrogen found 
by 13.72, in order to obtain an idea of the extent of albuminous 
destruction taking place in the body. If accurate results are desired, 
it becomes necessary to determine also the amount of nitrogen elimi- 
nated in the feces, a knowledge of the quantity in the food ingested 
being, of course, presupposed. 

With all these data given, the nitrogenous metabolism of the body 
can be accurately controlled. 

Example. — A patient eliminates 50 grams of urea in twenty-four 
hours; these 50 grams correspond to 50 X 13.72 — i. e., 686 grams 
of lean muscle tissue; on the other hand, he ingests an amount of 
nitrogenous material corresponding to only 10 grams of urea, equiva- 
lent to 10 X 13.72 — i. e., 137.2 grams of muscle tissue. The dif- 
ference between the amount ingested and that excreted in this case — 
i. e., 548.8 grams — must be referable to the destruction of organized 
albumin. 

When the amount of nitrogen eliminated is equivalent to that in- 
gested, nitrogenous equilibrium is said to exist. A healthy person is 
approximately in this condition. 

During starvation urea is still eliminated from the body, although 
in diminshed amount. The question now arises, What happens if 
at this time an amount of nitrogenous food is given which corresponds 
exactly in amount to that eliminated? Under such conditions an 
increased elimination of nitrogen takes place, all of the nitrogen 
ingested, in addition to that resulting from a breaking down of body 



CHEMISTRY OF THE URINE 403 

tissues, being excreted. The amount of nitrogen referable to the 
latter source, however, is somewhat less than that eliminated in the 
total absence of food. Unless starvation has been pushed too far, 
the body accommodates itself to the amount of food thus given and 
nitrogenous equilibrium is restored. If more food is allowed an 
increased elimination results, which again leads to a condition of 
nitrogenous equilibrium, different levels, so to speak, being possible. 

It is apparent, then, that the elimination of urea, and of nitrogen 
in general, is subject to great variation and depends to a great extent 
upon the amount ingested. A statement in figures expressing the 
daily elimination of urea and of nitrogen would, hence, be of very 
little value, especially in pathological conditions, in which the amount 
of nitrogen ingested is frequently very small. The elimination of 
nitrogen should hence always be compared with the amount ingested, 
for which purpose the tables of Konig 1 will be found most convenient. 
At the same time it must be remembered that not all the nitrogen 
taken into the body as food undergoes resorption, and that a variable 
amount, which in disease may be considerable, is eliminated with the 
feces, so that in accurate work this nitrogen also must be taken into 
account. In order to obviate the tedious estimation of nitrogen in the 
feces, it has been proposed to determine the standard amount of urea 
which should appear in the urine of a healthy person under different 
forms of diet. Such experiments, of course, presuppose the control 
person to be in a condition of nitrogenous equilibrium, which, from 
what has been said above, is readily accomplished, as the human body 
adapts itself with ease to different forms of diet. v In general practice, 
however, such a procedure would be difficult, but here approximate 
results can be obtained from a parallel estimation of the chlorides. In 
health the elimination of the chlorides may be placed at about one- 
half of the urea. Whenever the nitrogen resulting from tissue 
destruction is in excess of that referable to the proteids ingested, this 
relation between the excretion of chlorides and urea will be disturbed, 
as the tissues of the body contain very litte sodium chloride. When- 
ever the amount of urea is in excess of the normal amount of chlo- 
rides, as indicated above, an increased tissue destruction may be in- 
ferred, and vice versa. If, on the other hand, the chlorides are present 
in diminished amount, the conclusion may be drawn that a retention 
of albumins is taking place in the body; this is observed frequently 
during convalescence from acute febrile diseases. 

In most text-books the statement is found that the normal daily 
elimination of urea varies between 30 and 35 grams. This would 
imply that a lower amount could be viewed as abnormal. But, as 
I have pointed out, the urea elimination depends essentially upon the 
amount of proteid food ingested, and I have long maintained that the 

1 Chemie d. menschlichen Nahrungs u. Genussmittel, Berlin, 1893. 



404 THE URINE 

consumption of such large amounts of proteids as would lead to the 
elimination of the quantities stated is totally unnecessary. Every 
clinician no doubt can recall data which would tend to support this 
view, and Chittenden and Folin have demonstrated the same fact by 
numerous observations. As Folin says: the immediate elimination 
of the greater part of the nitrogen contained in 118 to 130 grams of 
proteid (Voit's standard) by means of the exogenous katabolism would 
seem to constitute very strong evidence in favor of the view that the 
proteid so katabolized can without harm, if not with advantage, be 
replaced by an equivalent quantity of carbohydrates. 

An increase in the amount of urea, and, as a matter of fact, of all 
the nitrogenous constituents, is observed especially in the acute 
febrile diseases, notwithstanding the diminished ingestion of nitrog- 
enous material, and is ascribed to the greatly increased tissue destruc- 
tion. 1 An excretion of 50 grams or more is here frequently observed. 
Formerly it was thought that the fever itself was responsible for 
this increased elimination. But this view became untenable when 
it was shown that the excretion of urea in the beginning of a febrile 
attack is not proportionate to the height of the temperature, reach- 
ing its higest point only when the fever has been continuous for several 
days. Still larger amounts, moreover, may be eliminated when the 
fever is abating. Similar observations have since been made. An 
increased elimination of nitrogen may also be noted in almost every 
case of ague preceding the onset of the fever. The latter, therefore, 
cannot be the only factor which causes the increased excretion of 
urea, and it has been suggested that the cells of the body have lost the 
power of taking up nitrogen. The question, however, whether this is 
dependent upon the increase in temperature or the action of certain 
toxic substances circulating in the blood, or upon both, still remains 
unanswered. 

The large increase in the elimination of nitrogen in febrile dis- 
eases is especially striking in those which end by crisis. This is 
notably the case in pneumonia, in which it may persist for two or 
three days after the occurrence of the crisis and is then no doubt 
largely due to the resorption of the exudate. 

Apparently, the only exception to the rule that the amount of 
urea is increased in acute febrile diseases is acute yellow atrophy, 
in which the excretion of urea is not only greatly diminished, but 
may cease altogether, its place being taken by other nitrogenous 
bodies, such as ammonium lactate, leucin, tyrosin, glycocoll, etc. 

Among afebrile diseases, in which an increased elimination of urea 
has been noted, may be mentioned the ordinary forms of diabetes 

1 Vogel, Zeit. f. rationelle Med., N. F., vol. iv, p. 362. Huppert, Arch. d. Heilk., 
vol. vii, p. 1. Lobisch, Wien. med. Presse, 1889, vol. xxxix, p. 1521. Huppert 
u. Riesellt, Arch. d. Heilk., vol. x, p. 329. Bauer u. Kunstler, Deutsch. Arch. f. 
klin. Med., vol. xxiv, p. 53. 



CHEMISTRY OF THE URINE 405 

mellitus, in which the highest figures have been obtained, viz., 150 
grams or more pro die. This is, in all probability, explained, in 
part at least, by the ingestion of excessive amounts of proteid food 
by such patients, but carefully conducted experiments seem to show 
that a not inconsiderable portion of the urea is directly referable to 
increased tissue destruction. The cases described by Hirschfeld * 
however, which will be considered later on, form an exception to 
this rule. 

v. Noorden and Lipm an- Wolff have shown that anemia as such 
is not necessarliy associated with a pathological increase in the albu- 
minous metabolism. But it appears that in pernicious anemia, at 
least in the bothriocephalus form, there are periods in which an in- 
creased albuminous disintegration does occur. According to Rosen- 
qvist, 2 this is far too extensive to be dependent entirely upon the 
destruction of red corpuscles, but must be associated with changes 
in other nitrogenous tissues of the body. After the expulsion of 
the worms a well-marked nitrogenous retention was observed. 
Similar results were obtained in cases of cryptogenetic pernicious 
anemia, where periods of markedly increased albuminous disinte- 
gration alternated sometimes with such of distinct nitrogenous reten- 
tion. Rosenqvist concludes that his observations are strongly in 
support of the theory that cryptogenetic pernicious anemia, like the 
bothriocephalus form, is also a toxic anemia. 

An unusually large output of nitrogen and greatly in excess of the 
amount ingested is apparently a common feature of acute leukemia. 
Ebstein records a case in which 62 grams of urea were eliminated 
in twenty-four hours, and Edsall 3 mentions an instance in which with 
an intake of only 7.25 grams of nitrogen, 29.534 grams appeared in 
the urine. 

In this connection it is interesting to note that an astonishing in- 
crease of the urinary nitrogen occurs on x-yslj treatment in those cases 
of chronic leukemia, when the characteristic response so far as the 
effect upon the spleen and the number of the leukocytes is concerned, 
takes place, while in the negative cases this is not observed. 4 

In purpura hemorrhagica a notable increase of the urinary nitrogen 
occurs, apparently without relation to the hemorrhages. Edsall men- 
tions an instance in which the patient, while ingesting not more than 
3 to 4 grams, eliminated amounts varying between 14 and 23 grams. 

A moderate increase has been found in severe cases of chronic leu- 
kemia, scurvy, minor chorea, and paralysis agitans. Observations 
made in cases of hystero-epilepsy have given rise to conflicting results. 

1 "Ueber eine neue klin. Form. d. Diabetes," Zeit. f. klin. Med., vol. xix, pp. 
294 and 325. 

2 Berlin, klin. Woch., 1901, vol. xxxviii, p. 666. 

3 Amer. Jour., Oct., 1905, p. 589. 

4 Edsall and Musser, Univ. of Penn. Med. Bull., Sept., 1905. 



406 THE URINE 

It is claimed, on the one hand, that the excretion of urea is diminished 
following convulsive seizures of a hystero-epileptic nature, in contra- 
distinction to an increased elimination following true epileptic attacks. 

In cases of functional albuminuria associated with an increased 
elimination of uric acid or oxalic acid, I have observed an increased 
elimination of urea, and believe that in the treatment of these diseases 
a systematic study of the excretion of nitrogen is of fundamental 
importance. The increase is here unquestionably due to the ingestion 
of excessive amounts of proteids. 

Of drugs, an increased elimination is produced by caffeine, mor- 
phine, codeine, ammonium chloride, sodium and potassium chlorides, 
lithium carbonate, following the ingestion of large amounts of water, 
etc. The data concerning the action of quinine, salicylic acid, cold 
baths, etc., are conflicting. A large increase has been observed in 
cases of phosphorus poisoning. 

Electricity appears to exert a marked influence upon the excretion 
of urea, producing an increased elimination. 

The diminished elimination of urea observed in certain diseases of 
the liver, 1 notably in acute yellow atrophy, carcinoma, cirrhosis, and 
even in Weyl's disease, is of special interest, and is in perfect accord 
with the theory that the liver is the main seat of its production. 

As has been stated, urea may disappear altogether from the urine 
in acute yellow atrophy and also in Weyl's disease, notwithstanding 
the frequently not inconsiderable degree of fever. In cirrhosis, 
hyperemia of the portal system has been thought to cause the dimi- 
nution, which may be increased further by the occurrence of ascites. 
In short, the factors which may be regarded as causing a diminished 
elimination of urea in hepatic diseases may be summarized under 
the following headings: 

1. Destruction of hepatic parenchyma. 

2. Diminished velocity of the flow of blood through the liver. 

3. Insufficient excretion of bile and coincident digestive disturb- 
ances. 

Whenever there is disease affecting that portion of the renal 
parenchyma which is concerned especially in the elimination of urea, 
a diminished amount will be met with, and carefully conducted 
observations upon the excretion of the various urinary constituents 
are here of considerable value from a diagnostic as well as a thera- 
peutic standpoint. However, as v. Noorden and others have pointed 
out, there are periods in the course of a nephritis when the urea 
output is quite normal. 

While, as a rule, the excretion of urea is greatly increased in dia- 

1 Hallerworden, Arch. f. exper. Path.u. Pharmakol., vol. xii. Weintraud, ibid., 
vol. xxxi. Stadelmann, Deutsch. Arch. f. klin. Med., vol. xxxiii. Fawitzki, ibid., 
vol. xlv. Frankel, Berlin, klin. Woch., 1878 and 1892. v. Noorden, Lehrbuch d. 
Path. d. Stoffwechsels, p. 287 



CHEMISTRY OF THE URINE 407 

betes mellitus, certain cases, which have been elaborately described 
by Hirschfeld, 1 must be excepted. His researches have established 
beyond a doubt that the resorption of nitrogenous material from the 
intestines may be very much below normal, and with it the elimina- 
tion of urea. Upon these grounds he has advocated the recognition 
of a distinct form of diabetes, which is characterized by a com- 
paratively rapid course, the occurrence of colicky abdominal pains 
before or at the onset of the diabetic symptoms proper, the existence 
of pancreatic lesions in a certain proportion of the cases, a more 
moderate degree of polyuria, etc. 

In mental diseases a diminished excretion of urea has been ob- 
served in melancholia and in the more advanced stages of general 
paresis, while an increase is associated with the increased ingestion 
of food during the first stage of profound dementia. 

Following epileptic, cataleptic, and hysterical seizures, as well as 
in pseudohypertrophic paralysis, a decrease has been noted by some 
observers. 

In tetanus the elimination of the urea nitrogen is normal or 
diminished. 

In Addison's disease a decrease is commonly noted. 

All forms of chronic, non-progressive anemia are associated with 
a decrease, as are also osteomalacia, impetigo, lepra, chronic rheu- 
matism, etc. In chronic lead poisoning the elimination of urea may 
be greatly diminished. Little is known of the influence of drugs in 
bringing about a diminished excretion of urea. * 

Properties of Urea. — Urea crystallizes in two forms, viz., in long, 
white needles if rapidly formed, or in long, colorless, quadratic 
rhombic prisms when allowed to crystallize gradually from its solutions. 

At 100° C. it begins to show signs of decomposition; at 130° to 
132° C. it melts; and when heated still further it is decomposed into 
cyanic acid and ammonia, of which the former is immediately trans- 
formed into its polymeric compound, cyanuric acid. Biuret is formed 
as an intermediary product during this decomposition, 2 molecules 
of urea yielding 1 molecule of ammonia and 1 molecule of biuret. 
As this substance, obtained on dissolving the residue remaining after 
all the ammonia has been driven off by careful heating, yields a 
beautiful reddish-violet color when a drop or two of a very dilute 
solution of cupric sulphate is added to its solution alkalinized with 
sodium hydrate, this reaction may be employed as a test in the detec- 
tion of urea (Biuret test). 

Very important is the behavior of urea when treated with a solu- 
tion of sodium hypochlorite or hypobromite, the most usual method 
of estimating urea being based upon this reaction, which may be 
represented by the equation 

CON 2 H 4 + 3NaOBr - 3NaBr + 2N + C0 2 + 2H 2 . 

1 Loc. cit. 



408 



THE URINE 



In the chapter on Reaction it was pointed out that urine gradually 
undergoes ammoniacal decomposition when exposed to the air; the 
ammonia is liberated from the urea according to the equation 

/HH 2 
CO( + H 2 = 2NH 3 + C0 2 . 

\NH 2 

This decomposition may also be effected by heating a watery solu- 
tion of urea in a sealed tube to 100° C. 

Urea is readily soluble in water, fairly so in alcohol, and insoluble 
in anhydrous ether and benzol. The aqueous solution of urea is 
neutral in reaction, but the substance combines with acids, bases, 
and salts to form molecular compounds. 

Urea nitrate, CON 2 H 4 .HN0 3 , crystallizes in two different forms: 
in thin rhombic or six-sided colorless plates, which are frequently 
observed arranged like shingles one on top of the other when rapidly 








Fig. 134. 



-Urea nitrate crystals. (Kruken- 
berg, after Kiihne.) 



Fig. 135. — Urea oxalate crystals 
berg, after Kufrne.) 



(Kruken- 



formed (Fig. 134), while larger and thicker rhombic columns or plates 
are obtained if the process is allowed to proceed more slowly. Urea 
nitrate is readily soluble in distilled water, while in alcohol and in 
water containing nitric acid it dissolves with difficulty. Upon heat- 
ing, it evaporates without leaving a residue. 

Urea oxalate, CON 2 H 4 .C 2 H 2 4 , crystallizes in rhombic or six-sided 
prisms or plates (Fig. 135), which are less soluble in water than the 
nitrate; in alcohol and in water containing oxalic acid it is only 
imperfectly soluble. 

With mercuric nitrate urea forms three different compounds, accord- 
ing to the concentration of the two solutions, viz., (CON 2 H 4 )Hg 2 (N0 3 ) 4 , 
(CON 2 H 4 ).Hg 3 (N0 3 ) 6 , and (CON 2 H 4 ) 2 .Hg(N0 3 ) 2 -f 3HgO. The lat- 
ter compound is of special importance, as Liebig's quantitative esti- 
mation of urea was based upon its formation. 



CHEMISTRY OF THE URINE 



409 



For the separation of urea from the urine the reader is referred to 
works on Physiological Chemistry. 

Quantitative Estimation of Urea. Hypobromite Method. — The 
method most commonly used in the clinical laboratory is the one 
based upon the decomposition of urea into carbon dioxide and nitro- 
gen in the presence of sodium hypobromite. The carbon dioxide 
thus formed is absorbed by an excess of sodium hydrate in the hypo- 
bromite solution, while the nitrogen is set free, and can be collected 
and measured; the determination 
of the corresponding amount of 
urea is then a simple matter. 

The hypobromite solution is pre- 
pared from two stock solutions. The 
first of these contain 125 grams of 
bromine and 125 grams of sodium 
bromide in 1000 c.c. of water. The 
second is a 22.5 per cent, solution 
of sodium hydrate. Immediately 
before use equal portions of the two 
solutions are mixed and diluted with 
one and one-half volumes of water. 

The reaction which takes place 
may be represented by the equation 

2NaOH + 2 Br = NaBr + NaOBr + H 2 .0 

Various forms of apparatus, 
termed ureometers, have been sug- 
gested for the estimation of urea 
by this method. One which I have 
found very " satisfactory is repre- 
sented in Fig. 136. It consists of 
a burette, C, with an ascending 
rubber tube attached to the reser- 
voir, B, which can be raised or 
lowered as required for the purpose 
of equalizing the pressure after col- 
lection of the gas. A descending 
tube leads to a wide-mouthed bot- 
tle, A, which contains the hypobromite solution. This is closed by 
a tightly fitting rubber stopper, to which a loop of platinum wire is 
attached carrying a little bucket made of glass or porcelain; this can 
be swung from its support by inclining the bottle. 

Method. — The rubber stopper is removed from the bottled, and 
water poured into B until the system B C is filled to such an extent 
that the water level is visible in B above the point where the rubber 
tube is attached. About 25 to 30 c.c. of the hypobromite solution 




tlllllllllllllllllllllllllllllHIIlllllllllllI 

riG. 136. —The author's ureometer. 



410 THE URINE 

are placed in the bottle A, and 2 c.c. of urine in the bucket; this is 
then attached to the wire loop. The stopper is now adjusted and 
the water in B and C brought to the same level, when the first read- 
ing is taken. A is then inclined until the bucket drops into the liquid 
below. The nitrogen which is liberated collects in the burette C; as 
a consequence the water falls in C and rises in B. After twenty to 
thirty minutes the pressure in C is equalized by lowering B until the 
water in both tubes is at the same level. The second reading is then 
taken, the difference between the two indicating the volume of nitro- 
gen liberated from 2 c.c. of urine at the temperature of the water in 
C B, which, as well as the barometric pressure, should be previously 
noted. 

As the volume of gases is influenced by the temperature, the baro- 
metric pressure, and the tension of the aqueous vapor, it becomes 
necessary, in order that the results reached shall be comparable with 
those obtained by other observers, to reduce the volume of nitrogen 
actually noted to a certain standard. This has-been placed at 0° C 
and 760 mercury millimeters pressure, in the absence of moisture. 
The correction is made according to the following formula: 

V = 7 fl n /-i _i n on^fifi tV * n wn * cn ^ represents the corrected 

volume of the gas in terms of c.c, v the volume actually observed, 
B the barometric pressure in Hgmm., T the tension of the aqueous 
vapor at the temperature noted, t. The volume of nitrogen observed 
being thus corrected, the calculation of the corresponding amount 
of urea is based upon the following considerations : From the formula 
CON 2 H 4 it is apparent that 2 atoms of nitrogen are contained in 1 
molecule of urea; in other words, that 28 parts by weight of nitrogen 
correspond to 60 parts by weight of urea. The equivalent of 1 gram 
of urea is then found according to the equation: 60 : 28 : : 1 : x; and x 
=0.46666. The volume corresponding to 0.4666 gram of dry nitrogen 
at 0° C. and 760 Hgmm. pressure is 372.7 c.c. It has been found, 
however, that only 354.3 c.c. of nitrogen are evolved from 1 gram of 
urea at best when the hypobromite method is employed Knowing 
that 354.3 c.c. of nitrogen correspond to 1 gram of urea, the amount 
of urea to which the volume of nitrogen actually observed is refer- 
able would then be found according to the equation 

y 
1 : 354.3 : : x : y; and x= ' . 5 , in which y denotes the number of 

cubic centimeters of nitrogen evolved from 2 c.c. of urine, and x the 
corresponding amount of urea. In order to ascertain the percentage 
amount of urea it is only necessary to multiply the figure just obtained 
by 50. 

Precautions: (1) The urine must be free from albumin. (2) It 
should contain only about 1 percent, of urea — i. e., not more than 0.025 



CHEMISTR Y OF THE URINE 



411 



gram in 2 c.c. Whenever a greater amount is noted, therefore, the 
urine is. diluted to the proper degree, due allowance being made in 
the calculation. 

In ordinary clinical work the barometric pressure, as well as the 
tension of the aqueous vapor, may be ignored. 

Of the many other ureometers the one devised by Doremus in the 
modification of Heinz is most convenient and furnishes \^ery satis- 
factory results. 



rr\ 





Fig. 137. — Doremus-Heinz ureometer. 



Fig. 138.— Folin's safety-tube. 



Its general construction is seen in Fig. 137. A small amount of 
urine is poured into B while the stopcock (C) is closed. This is then 
opened for a moment and again closed, so as to fill its lumen. The 
tube A is washed out with water and filled with the hypobromite 
solution. The tube B is filled with urine to the zero mark, and 1 c.c. 
(or less, if the urine is concentrated) is allowed to mix with the hypo- 
bromite solution 1 in A. After all bubbles of gas have disappeared 
the reading is taken. Each small division corresponds to 0.001 gram 



This is prepared as described on p. 409. 



412 THE URINE 

of urea and every ten divisions hence to 0.01 gram, for the amount of 
urine used. 

The urine must be free from albumin and should not contain more 
than 1 per cent, of urea. If necessary it is diluted with water. 

In the presence of ammonium compounds the results may be faulty, 
and in cases where this is suspected it is advisable to resort to more 
accurate methods, such as that of Folin. 

Method of Folin. 1 — This is based upon the following considera- 
tions: At a temperature of about 160° C. crystallized magnesium 
chloride, MgCl 2 .6H 2 0, boils in its water of crystallization. In such 
a solution urea is quantitatively decomposed into ammonia and car- 
bon dioxide within one-half hour. If the process is carried out in 
acid solution, the ammonia can subsequently be distilled off after 
rendering the mixture alkaline, and is then titrated. The correspond- 
ing amount of urea is ascertained by calculation. At the same time, 
however, the preformed ammonia is obtained, and it is hence necessary 
to eliminate this source of error by a separate estimation of this form. 
This is conveniently done according to the method which has like- 
wise been suggested by Folin (see below). 

Method. — 3 c.c. of urine when carefully measured with a 5 c.c. 
pipette graduated in twentieths are placed in an Erlenmeyer flask 
of 200 c.c. capacity, together with 20 grams of magnesium chloride 
and 2 c.c. of concentrated hydrochloric acid. (The magnesium chlo- 
ride usually contains a small amount of ammonia, which must be 
separately determined.) The flask is closed with a perforated stopper 
through which a specially constructed safety-tube passes (see Fig. 
138). 2 The mixture is now boiled until the drops flowing back 
through the tube produce a hissing sound on coming in contact with 
the solution. After this point has been reached the boiling is con- 
tinued more moderately for about forty-five minutes. Immoderate 
foaming during this process and the subsequent distillation is guarded 
against by adding a small piece of paraffin (about the size of two coffee 
beans). 

The solution while still quite hot is carefully diluted to about 500 
c.c. — at first by allowing the water to flow drop by drop through the 
tube; it is then transferred to a 1000 c.c. retort, treated with about 7 
or 8 c.c. of a 20 per cent, solution of sodium hydrate, and the ammonia 
distilled off into a measured amount of a decinormal solution of sul- 
phuric acid. The distillation may be interrupted when about 350 
c.c. have passed over (viz., after about sixty minutes). The distillate 
is boiled for a moment to remove any carbon dioxide which may be 
present in solution, and on cooling is titrated to determine the excess 
of acid. Each cubic centimeter of the decinormal ammonia present 

1 Zeit. f. physiol. Chem., vol. xxii, p. 504, and vol. xxxvi, p. 333. 

2 The tube can be obtained from Messrs. Eimer and Amend, of New York. 



CHEMISTRY OF THE URINE 



413 



in the distillate corresponds to 0.003 gram, viz., to 0.1 per cent, of 
urea. 

From this result the amount of preformed ammonia and that 
present in the 20 grams of magnesium chloride must be deducted. 

Estimation of Nitrogen. — For the purpose of estimating the total 
amount of nitrogen in the urine, the method of Kjeldahl is most con- 
veniently employed. 

Kjeldahl's Method. 1 Principle. — The organic matter of the urine 
is decomposed by means of sulphuric acid, when all the nitrogen 




Fig. 139. — Kjeldahl's nitrogen apparatus 



which is not present in combination with oxygen is transformed into 
ammonia. After adding sodium hydrate in excess the ammonia 
is distilled off and received in a known quantity of titrated acid, 
the excess being retitrated with sodium hydrate. In this manner 
the amount of ammonia and the corresponding quantity of nitrogen 
are ascertained, it being remembered that 17 grams of ammonia 
correspond to 14 grams of nitrogen. 

Reagents required: 

1. Gunning's mixture. This consists of 15 c.c. of concentrated 
sulphuric acid, 10 grams of potassium sulphate, and 0.5 gram of 

1 "Neue Methode zur Bestimmung des Stickstoffes in organischen Korpern," 
Zeit. f. analyt. Chem., 1883, vol. xxii, p. 366. 



414 



THE URINE 



cupric sulphate. In the place of Gunning's mixture one of 500 c.c. 
of concentrated sulphuric acid and 100 grams of phosphoric anhy- 
dride may also be employed, and has the advantage that oxidation 
proceeds more rapidly. 

2. A solution of sodium hydrate containing 270 grams in the liter 
(sp. gr. 1.243). 

3. Pulverized talcum or granulated zinc. 

4. A one-fourth norma' solution of sulphuric acid. 

5. A one-fourth normal solution of sodium hydrate. 
Apparatus required (see Fig. 139): This consists of a retort of 

about 750 c.c. capacity (A), which is connected with a Kjeldahl 

distilling tube (B), and through this 
with a Stadeler condenser (C). The 
ammonia is received in the nitrogen 
bulb at D. In addition a Kjeldahl 
digesting flask of 200 to 300 c.c. 
capacity is required. 

Method. — Five or 10 c.c. of urine 
are placed in the digesting flask and 
treated with Gunning's mixture. To 
this end it is best to add the sulphuric 
acid and cupric sulphate first, to heat 
until sulphuric acid vapors are given 
off in abundance, and then to add 
the potassium sulphate. The heat- 
ing is continued until the solution 
becomes entirely clear and almost 
colorless, the flask being inclined 
at an angle of about 45 degrees. 
Vigorous ebullition should be avoided. 
If the sulphuric acid-phosphoric anhydride mixture is to be employed, 
the urine is first treated with 0.4 gram of mercuric oxide, and 10 c.c. 
of the acid mixture added. Digestion is then carried on as described. 
Toward the end of digestion, in either case, it is advantageous to 
throw a few crystals of potassium permanganate into the fusion, so 
as to ensure complete oxidation. 

Upon cooling, the contents of the flask are transferred to the 
retort with the aid of a little water, and slowly treated with a moder- 
ate excess of the sodium hydrate solution. As a general rule, 40 
c.c. for each 5 c.c. of sulphuric acid are sufficient. A little pulver- 
ized talcum or a few pieces of granulated zinc are finally added; the 
retort is connected with the condenser with the interpositon of the 
distilling tube and the distillation begun. The talcum or zinc serves 
the purpose of preventing undue frothing and bumping. The dis- 
tillation is continued until about two-thirds of the solution have passed 
over. The distillate is received in the nitrogen bulb, which should 




Fig. 140. — Kjeldahl's apparatus for the 
simultaneous oxidation of six specimens: 
o, Kjeldahl flasks. 



CHEMISTRY OF THE URINE 



415 



contain a carefully measured quantity of the one-fourth normal 
solution of sulphuric acid. As a general rule, 30 c.c. are sufficient. 
As soon as the distillation is completed the condenser is discon- 
nected, washed out with a small amount of distilled water, and the 
washings added to the distillate. After the addition of a few drops 
of tincture of cochineal or dimethyl-amino-azo-benzol the excess 
of sulphuric acid is retitrated with the one-fourth normal solution 
of sodium hydrate, and the amount found deducted from the 30 c.c. 
used. The titration should be continued until every trace of yellow 
(in the case of the cochineal) has disappeared and a pure rose color is 




Fig. 141. — Kjeldahl's apparatus for the simultaneous distillation of six specimens: 
a, condenser; b, -distillation flasks; c, receivers. 

obtained, or, in the case of the dimethyl-amino-azo-benzol, until the 
last trace of red has disappeared and the solution has turned yellow. 
The difference multiplied by 0.0035 will indicate the amount of 
nitrogen present in the 5 or 10 c.c. of urine. The corresponding 
amount of urea is found by multiplying this figure by 20. 

Whenever several nitrogen determinations are to be carried out 
daily it is convenient to make use of a special apparatus, which per- 
mits of such determinations being conducted at one time. The 
general plan of the outfit is seen in the accompanying illustrations 
(Figs. 140 and 141). 

Ammonia. 



Every urine contains a small amount of ammonia, which normally 
varies but little, and corresponds to from 4.1 to 4.64 per cent, of the 
total amount of nitrogen, viz., to about 0.7 gram in the twenty- 
four hours. It is present in combination with the various acids of 
the urine, and in all likelihood represents a small amount of the 



416 THE URINE 

ammonia which has not been transformed into urea, but has been 
utilized to saturate the affinities of a slight excess of acid, formed 
during the nitrogenous metabolism of the body, over the available 
fixed alkalies. 

In man an increased elimination of ammonia is observed when- 
ever an increased formation of acids occurs, or whenever a sufficient 
supply of oxygen is not available. In the latter case, no doubt, 
the increased elimination is owing to the fact that in consequence 
of the deficient supply of oxygen the synthetic formation of urea 
is impeded in the liver. As this organ, moreover, is the principal 
seat of the synthesis of urea, we can readily understand that extensive 
parenchymatous degeneration, as in acute yellow atrophy, in phos- 
phorus poisoning, etc., will lead to an increased elimination of 
ammonia. 

In any event, the relative increase of the ammonia is the essential 
factor, while variations in its absolute quantity are of secondary 
importance. Some of the results which have been obtained in various 
diseases are given in the following table: 

Per cent. 

Normal values 4.10- 4.64 

Febrile diseases 5.72- 6.70 

Carcinoma of the liver 6.40-24.50 

Liver abscess (actinomycosis) 10.60 

Circulatory dyspnea 13.10-32.20 

Respiratory dyspnea 6.60-14.30 

Abnormally high absolute values are quite constantly observed in 
diabetes, in which a daily elimination of from 4 to 5 grams may be 
regarded as common. In a general way the amount of ammonia in 
cases of diabetes gives an idea of the amount of organic acids; but, 
as Herter has pointed out, we cannot detect moderate quantities of 
organic acids in this way. (See Oxybutyric Acid.) 

In cases of pernicious vomiting of pregnancy Williams 1 found a 
large increase of ammonia, up to 20 to 45 per cent. , while this does 
not occur in nervous vomiting and in eclampsia. It is advised that 
in such cases the uterus be emptied, when the ammonia is said to 
drop at once. 

A slight rise occurs also in normal pregnancy and reaches its maxi- 
mum during labor. 

Very curiously a diminished elimination of ammonia is observed 
in many cases of nephritis so long as symptoms of venous stasis do 
not exist. 

In a case of pernicious anemia relative amounts, varying between 
3.3 and 5.6 per cent., were obtained during the days immediately 
preceding death. 

Quantitative Estimation. Folin's Method. — 10 c.c. of urine are 
diluted to about 45 c.c, treated with a small amount of burnt mag- 

] Amer. Jour, of Med. Sci., September, 1906. 



CHEMISTRY OF THE URINE 



417 



nesia (0.5 gram), and boiled for forty-five minutes, the distillate being 
received in decinormal sulphuric acid through an absorption tube, 
such as the one pictured in Fig. 142. This consists of a glass tube, 
a, measuring about 8 mm. in diameter, one extremity of which has 
been blown into the small bulb b. By means of a heated platinum 
wire five or six holes, each about 1 mm. in diameter, are made in the 
bulb ; c is a rubber stopper which fits into the second 
tube d. This is merely a test-tube (2.5 cm. in diam- 
eter) which has been cut about 7.5 cm. from the 
upper end. About 3 cm. from the upper margin 
this tube is provided with six or seven holes as in 
bulb b. The entire apparatus is directly immersed 
in the decinormal acid and ensures the complete 
absorption of the ammonia in one flask, even if this 
contains only 5 to 10 c.c. of the acid. The ammonia 
is then determined by titration as above, using 
alizarin red as indicator; 2 drops of a 1 per cent, 
solution suffice for 200 to 300 c.c. The titration is 
carried to the red point, not to the violet. As a 
small amount of urea, however, is decomposed during 
the prolonged ebullition, it is necessary to ascertain 
separately the quantity of ammonia which is referable 
to this source. To this end the retort is opened at 
the expiration of forty-five minutes, and an amount 
of water added which is approximately equivalent to 
that of the distillate. The distillation is then con- 
tinued for another period of forty-five minutes; the 
distillate is received in decinormal sulphuric acid, 
and the ammonia referable to decomposition of the 
urea estimated as before. The difference between the 
two results indicates the amount of preformed ammonia that was 
originally present; 1 c.c. of the y^- sulphuric acid indicates 0.0017 
gram of ammonia. 

This method is also applicable for the determination of ammonia 
in the blood. 




Fig. 142.— Absorp- 
tion tube. 



Literature. — Hallervorden, Arch. f. exper. Path., vol. xii, p. 237. Stadol- 
mann, Deutsch. med. Woch., 1889, p. 942. Michaelis, ibid., 1900, p. 276. O. 
Folin, Zeit. f. physiol. Chem., vol. xxxii, p. 575, and ibid., 1902, vol. xxxvii, 
p. 161. 



Uric Acid. 



According to our present views, uric acid, in man, is not formed 
during the decomposition of all albuminous substances, as was for- 
merly supposed, but constitutes a specific product of decomposition 

27 



418 THE URINE 

of one class of albumins only, namely, the nucleins. 1 It appears, 
moreover, that the mother-substance of uric acid is confined to the 
true nucleins, viz., to those containing a nucleinic acid radicle, 
while the paranucleins, in which this is lacking, are without effect 
upon the elimination of uric acid. On decomposition the nucleins 
give rise to the appearance of the xanthin, alloxuric, or purin bases, 
which on oxidation are transformed to uric acid. According to Emil 
Fischer, 2 the xanthins are derived from an hypothetical compound 
which he terms purin, and which he supposes to be constituted as 
shown in the formula 

(6) 

(1)N— CH 

I I (7) 

(2)HC (5)C NH 



(3)N C Nc: 

(4) (9) 



:CH(8). 



By substituting the group NH 2 for the H atom at 6, adenin thus 
results, and is hence also spoken of as 6-aminopurin: 

N C.NH 2 

I I 

HC C NH X 

II II X CH. 

N^ — C N^ 

Hypoxanthin, according to this conception, would be 6-oxypurin; 
xanthin 2, 6-dioxypurin, and guanin 2-amino-6-oxypurin, as shown 
by the structural formulas: 

HN CO HN CO 

II II 
HC C NH X CO C NH X 

II II icH. I II >CH. 

N C N^ HN C N^ 

Hypoxanthin. Xanthin. 

NH CO 

I I 
HN-C C NH X 

i II J^CH. 

HN C N^ 

Guanin. 

From the structural formula of purin it is also apparent that still 
other derivatives of this substance may exist, and as a matter of 
fact others are known, viz., mono-methylxanthin or heteroxanthin, 
di-methylxanthin or paraxanthin, tri-methylxanthin, the isomeric 
compounds of paraxanthin, viz., theophyllin and theobromin, and 
others. Their relation to xanthin is shown in the formulas: 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co. 

2 Jier. d. Deutsch. chem. Ges., 1897, vol. xxx, p. 54$. 



CHEMISTRY OF THE URINE 



419 




HN CO 

I I 
CO C N.CH 3 > 

I II 

HN C N 

Heteroxanthin. 



CH 3 .N CO 

I i 



NH 



CH. 



I I! J^CH. 

CH 3 .N C N=^ 

Theophyllin. 

CHo.N CO 

I I 
CO C N.CH, 

I II 

CH3.N C N: 

Caffeine. 



Two of these bodies, namely, heteroxanthin and paraxanthin, have 
also been found in the urine. 

From these basic substances, then, which are found in the nucle- 
inic acid radicle of the true nucleins, uric acid is supposedly 
derived, and there are numerous facts which go to show that this 
supposition is in all likelihood correct. It will thus v be observed 
that structurally uric acid is intimately related to the bodies in ques- 
tion, and, like these, contains the purin radicle: 



HN CO 

I I 
CO C NH 

I II 

HN C NH 

Uric acid. 



CO. 



It may hence be regarded as 2, 6, 8 tri-oxypurin. Uric acid and 
the xanthin bases, moreover, qualitatively, all yield the same decom- 
position products when treated with fuming hydrochloric acid or 
hydriotic acid under high pressure; only the quantitative relations 
vary, as shown in the equations: 

C 5 H 5 N 5 + 8H 2 = 4NH 3 + C0 2 + CH 2 .NH 2 .COOH + 2H.COOH. 
Adenin. Glycocoll. Formic acid. 

C 5 H 4 N 4 + 7H 2 = 3NH 3 + C0 2 + CH 2 .NH 2 .COOH + 2H.COOH. 

Hypoxanthin. 

C 5 H 5 N 5 + 7H 2 = 4NH 3 + 2C0 2 + CH 2 .NH 2 .COOH + H.COOH. 
Guanin. 

C 5 H 4 N 4 2 + 6H 2 = 3NH 3 + 2C0 2 + CH 2 .NH 2 .COOH + H.COOH. 
Xanthin. 

5 H 4 N 4 O 3 + 5H 2 = 3NH 3 + 3C0 2 + CH 2 .NH 2 .COOH 
Uric acid. 



In accordance with this supposed origin of uric acid we find an 
increased elimination following the ingestion of all substances which 
contain purin bases either as such or in the form of true nucleins 



420 THE URINE 

(endogenous uric acid). At the same time it must be remembered 
that uric acid may also result from the nucleins of the body tissues; 
and we find, as a matter of fact, that during starvation uric acid does 
not disappear from the urine (endogenous uric acid). The principal 
source of the uric acid under such conditions are the nucleins of the 
leukocytes; and, according to Horbaczewski 1 and others, this source 
is indeed more important than the nucleins of the food. According 
to this idea, the latter call forth an increased elimination of uric acid 
only in an indirect manner — i. e., by stimulating more strongly than 
other food-stuffs the cell formation and cell destruction of the body. 
However this may be, there can be no doubt that the amount of uric 
acid eliminated in the urine depends, in the first instance, upon the 
amount of nucleins or purin bases as such which are ingested, and 
upon the degree of nuclear destruction which takes place in the body. 
Other factors, however, also enter into consideration. We thus know 
that the body is capable of transforming a certain amount of uric acid 
into urea. This fact was pointed out long ago by Frerichs and 
Wohler, and has recently again been confirmed. It was found that 
after the ingestion of large amounts of nucleins only a certain por- 
tion of the nuclear nitrogen is eliminated as uric acid, and that this 
amount is extremely variable. Whether individual peculiarities 
have any part in determining this amount is unknown, but not 
improbable. Oxidation on the part of the body tissues must also 
be taken into consideration, and it unquestionably varies not only 
in different people, but also in the same individual at different times. 
Then again there is evidence to show that under certain conditions 
uric acid may be formed synthetically in the body. That this is the 
usual mode of formation in birds and reptiles has been shown by 
Minkowski, 2 who found that after extirpation of the liver in geese 
the greater portion of the urinary nitrogen was eliminated in the 
form of ammonia in combination with lactic acid. In the human 
being very little uric acid is in all likelihood formed in this manner 
under normal conditions, but the possibility of its occurrence, in 
disease more particularly, should not be overlooked. As uric acid, 
moreover, may in part at least be eliminated in the feces, it is clear 
that the amount which appears in the urine cannot be regarded as an 
accurate index of the degree of nuclear destruction or of the amount 
which is formed in the body tissues. That retention of uric acid 
can further occur in the body, which may or may not be followed 
by increased elimination, is likewise undoubted. 

According to our present knowledge, uric acid is formed in all the 
organs of the body, including the bone-marrow, the muscles, the 

1 Beitrage zur Kenntniss der Bildung von Harnsaure, etc., Monatshefte fur 
Chem., 1891, vol. xii, p. 221; and Wien. Sitzungsber, vol. c. 

2 "Ueber den Einfluss d. Leberextirpation auf den Stoffwechsel," Arch. f. 
exper. Path u, Pharmakol., 1886, vol. xxi, p. 41, 



CHEMISTRY OF THE URINE 421 

spleen, the liver, the kidneys, etc. Under pathological conditions it 
may also originate in the joints and tendons. 

Under normal conditions the daily elimination of uric acid varies 
between 0.2 and 1.5 grams, thus constituting -^ to y^ part of 
the total urinary nitrogen. It is largely influenced by the character 
of the diet, the amount of exercise taken, the general health of the 
individual, etc. After the ingestion of large amounts of food rich 
in nucleins, such as thymus gland, liver, kidneys,and brain, a corre- 
sponding increase in the amount of uric acid is observed. Generally 
speaking, animal food causes a greater elimination of uric acid than 
vegetable food, and it is supposed that this difference is essentially 
due to the extractives of the meat. 1 Of special interest is the increase 
in the elimination of uric acid which is observed five hours after the 
ingestion of a full meal. This increase, according to Horbaczewski, 2 
is associated with the disappearance of the digestive leukocytosis and 
consequent leukolysis. 

Some observers have attached much importance to the relation exist- 
ing between the elimination of uric acid and urea, and are inclined to 
assume the existence of a special uric acid diathesis when this rela- 
tion continuously exceeds the usual standard of 1 to 50, or 1 to 60. 
This question is an extremely intricate one, and we are scarcely 
in a position to speak definitely of the significance of such varia- 
tions. On the one hand, there can be no doubt that an unusually 
high uric acid coefficient may be met with in individuals who are 
apparently in good health, while in others, in whom larger actual 
amounts of uric acid are eliminated than are usual, normal or even 
subnormal values may be found. The entire question of the uric 
acid diathesis is in a chaotic condition, and it would perhaps be 
well to speak of such a diathesis only when a distinct increase is 
continuously observed. That numerous symptoms of a neurasthenic 
type are often seen when the uric acid coefficient is increased is a 
matter of daily observation, but it would be premature to regard this 
symptom as a causative factor of the disease in question. 3 Even in 
gout it can scarcely be said that uric acid has been proved the materia 
peccans, and our knowledge concerning the etiology of the disease is 
still as obscure as when Garrod 4 showed that an accumulation of uric 
acid occurred in the blood of such patients. Hitherto it has been 

1 A Hermann, "Abhangigkeit der Harnsaureausscheidung von Nahrungs- und 
Genussmitteln," Deutsch. Arch. f. klin. Med., 1888, vol. xliii, p. 273. See also 
W. Camerer, Zeit. f. Biol., N. F., 1896, vol. xv, p. 140. 

2 Harnsaureausscheidung u. Leucocytose, Sitzungsber. d. Wiener Akad. d. 
Wissensch., 1891, Abth. 3. See also Lowit, Studien z. Physiol, u. Path. d. Blutes, 
1892. W. Kiihnau, " Das Verhaltniss d. Harnsaureausscheidung zur Leucocy- 
tose," Zeit f. klin. Med., vol. xxviii, p. 534. P. F. Richter, " Ueber Harnsaure- 
ausscheidung und Leucocytose," ibid., vol. xxvii, p. 290. 

3 C. E. Simon, Amer. Jour. Med. Sci., 1899, p. 139, andN. Y. Med. Jour., 1895, 
p. 330. 

4 On the Nature and Treatment of Gout, 1847. 



422 THE URINE 

supposed that the deposition of urates in the joints and periosteum 
of gouty patients is referable to a diminished alkalinity of the blood, 
and that acute paroxysms result whenever an increase in its alkalinity 
occurs, leading to a resorption of the urates previously deposited and 
a consequent flooding of the system with the material in question. 
As a matter of fact, a considerable diminution in its excretion is 
observed immediately preceding the attack, while during the par- 
oxysm and immediately following it a corresponding increase is noted. 
Numerous investigations, however, have shown that distinct changes 
in the alkalinity of the blood do not occur in gout, and that an increase 
in the amount of uric acid in the blood may not only be observed in this 
disease, but in other diseases as well which are not associated with 
gouty symptoms. The conclusion is hence justifiable that the pres- 
ence of uric acid in the blood per se cannot be offered as an explana- 
tion of the occurrence of a gouty attack. 1 Futcher, 2 who has recently 
observed a number of cases of gout with modern methods, states that 
he almost invariably found that before the onset of the acute symp- 
toms the uric acid is below and often far below 0.4 gram. On 
the second or third day after the beginning of the acute symptoms 
the uric acid curve steadily rises, reaching 0.8 to 1.9 grams or even 
higher values. With the subsidence of the acute symptoms the 
curve gradually falls below the lower limit of the normal, and in 
the interval between the acute attacks the excretion may be only 0.1 
to 0.2 gram daily. In one very marked chronic case Futcher found 
no uric acid excretion whatever on certain days during the interval. 
The phosphoric acid curve runs a course almost parrellel to that 
of the uric acid, which suggests quite strongly that even in gout the 
uric acid is derived from nucleins, and is not formed synthetically, 
as might possibly be imagined. 

The greatest increase in the elimination of uric acid is observed 
in leukemia, in which the quantity may amount to over 12 grams in 
the twenty-four hours (case of Magnus-Levy). That the increased 
elimination in this disease is referable to the enormous increase in the 
number of the leukocytes and consequent leukolysis can scarcely be 
doubted. In other diseases which are associated with a high grade 
of leukocytosis, and especially those in which the disease terminates 
by crisis or hastened lysis, such as erysipelas and pneumonia, a con- 
siderable increase is likewise observed, and is referable to the same 
origin. This increase is especially marked immediately after crisis 
has occurred, but it not infrequently precedes this by several hours. 

1 B. Laquer, Ueber die Ausscheidungsverhaltnisse der Alloxurkorper. Berg- 
mann, 1906. (Full literature.) C. von Noorden, Lehrbuch d. Pathologie d. 
Stoffwechsels, Berlin, 1893. W. Ebstein, "Die Natur u. Behandlung der Gicht," 
Verhandl. d. VIII Congr. f. inn. Med., 1889, p. 133. 

2 " The Occurrence of Gout in the United States," Jour. Amer. Med. Assoc, 
1902, vol. xxxix, p. 1046. 



CHEMISTRY OF THE URINE 423 

In the other febrile diseases an absolute increase is less marked and 
inconstant. 

In diabetes a diminished amount of uric acid is usually found. 
Cases may be seen, however, in which, associated with a diminution 
or an entire disappearance of the sugar, a most marked increase occurs, 
amounting in some cases to 3 grams in the twenty-four hours. To 
this condition the term diabetes alternans has been applied. 

In acute articular rheumatism an increased elimination is observed 
so long as the temperature remains high, while with approaching 
convalescence the amount returns to normal, and may even fall 
below normal. In chronic rheumatism, on the other hand, no con- 
stant relations have been observed. 

In the ordinary forms of anemia and chlorosis the amount of 
uric acid is quite constantly diminished, as also in chronic inter- 
stitial nephritis, chronic lead poisoning, progressive muscular atro- 
phy, and pseudohypertrophic paralysis. 

According to Krainsky, Haig 1 and Caro, 2 a decrease in the output 
of uric acid precedes the epileptic attack, and is subsequently followed 
by a rise to the same degree. Haig also noticed this in connection 
with attacks of migraine. 

Rather low amounts are reported by Edsall in a case of purpura 
hemorrhagica. 

Of special interest is the observation by Edsall that in those cases 
of chronic leukemia in which there is a response to avray treatment 
uric acid and purin bases are at once markedly increased. 

Properties of Uric Acid. — The close relation existing between uric 
acid and the xanthin bases has been already considered. By oxida- 
tion uric acid is transformed into urea or into substituted ureas, 
such as allantoin and alloxan, which latter in turn is closely related 
to parabanic acid or oxalyl-urea and barbituric acid or malonyl urea. 

Pure uric acid forms a white, crystalline powder which is almost 
insoluble in cold water (1 to 40,000), with difficulty soluble in boiling 
water (1 to 1800), and insoluble in alcohol and ether. In concentrated 
sulphuric acid it dissolves with ease, but is precipitated upon dilu- 
tion with water. In aqueous solutions of the alkaline carbonates 
and hydrates it dissolves, with the formation of neutral or acid salts, 
as represented by the equations: 

C 5 H 4 N 4 3 + Na 9 C0 3 =C 5 H 3 NaN 4 3 + NaHC0 3 . 
C 5 H 4 N 4 3 + 2Na 2 C0 3 = C 5 H 2 Na 2 N 4 3 + 2NaHC0 3 . 

In freshly voided urine uric acid is said to occur as a quadriurate, 
viz., as a compound in which one molecule of sodium is in combina- 
tion with two molecules of uric acid. The quadriurate, however, is 

1 Brain, 1896, p 191. 

2 Deutsch. med. Woch., 1900, No. 19. 



424 THE URINE 

readily decomposed with the formation of uric acid and acid urates 
(biurates). Its solubility in the urine depends upon the amount of 
water present, the reaction, and the presence of inorganic salts. 
When acid sodium phosphate preponderates, the biurate is precipi- 
tated, while free uric acid is thrown down when disodic phosphate 
only is present. Neutral urates cannot occur in the urine. The 
basic substances which may occur in the urine in combination with 
uric acid are sodium, potassium, ammonium, and possibly also 
calcium and magnesium. These salts may be decomposed by the 
addition of a sufficiently large quantity of a stronger acid, such as 
hydrochloric acid, when uric acid is set free. The acid salts are 
soluble with great difficulty, and are hence precipitated whenever the 
urine is markedly acid or concentrated, and also when it is exposed 
to a low temperature. This holds good especially for the acid 
ammonium compound, and upon this fact FolhVs quantitative 
estimation of uric acid is based. 

Pure uric acid crystallizes in transparent, colorless, rhombic plates, 
while that which usually separates from the urine is of a reddish- 
brown color and may assume a great variety of forms (Fig. 143). 
Of these, the so-called whetstone form is the most characteristic (see 
Sediments). Colorless rhombic platelets may, however, also be seen. 

Of the compounds which uric acid forms with the heavy metals, 
the silver salt is especially important. When a solution of uric acid 
in ammonia is treated with an ammoniacal solution of silver nitrate 
(see below) the solution remains clear; but if calcium chloride, 
sodium chloride, or magnesia mixture is then added, a precipitate 
forms, which contains the uric acid in combination with silver. 

Test for Uric Acid. Murexid Test. — A few crystals are dissolved 
by means of a few drops of concentrated nitric acid, with the appli- 
cation of heat, upon a porcelain plate, such as the cover of a crucible. 
The nitric acid is then carefully evaporated, when a yellowish-red 
spot will remain. Upon cooling, a drop of ammonia is placed upon 
this spot, when in the presence of uric acid a beautiful purplish-red 
color develops, owing to the formation of ammonium purpurate 
(murexid). If now a drop of sodium hydrate solution is added, the 
color changes to a reddish blue, which disappears upon heating; the 
reaction thus differs from the somewhat similar xanthin reaction. 

Folin's Modification of Hopkins' Method. 1 — This is the most con- 
venient method for the estimation of uric acid in the urine, and as 
accurate as the more complicated procedure of Ludwig-Salkowski. 
It is based upon the precipitation of uric acid by ammonium sulphate, 
as ammonium urate, the decomposition of the latter by sulphuric 
acid, and the estimation of the liberated uric acid by titration with 
potassium permanganate. To precipitate the uric acid, and also to 

1 O. Folin u. A. Shaffer, Zeit. f. physiol. Chem., vol. xxxii, p. 552. 



CHEMISTRY OF THE URINE 



425 



remove the small amount of mucoid substance which is found in 
every urine, the following reagent is employed : 500 grams of ammo- 
nium sulphate and 5 grams of uranium acetate are dissolved in 650 c.c. 
of water, to which solution 60 c.c. of a 10 per cent, solution of acetic 
acid are further added. The resulting solution measures about 1000 
c.c; 75 c.c. of the reagent are added to 300 c.c. of urine in a 
flask holding 500 c.c. After standing for five minutes the mixture 




Fig. 143. — Uric acid crystals. 

is filtered through two folded filters, and thus freed from the mucoid 
body, which is carried down with the uranium phosphate in acid solu- 
tion. The filtrate is divided into two portions of 125 c.c. each, which 
are placed in beakers and treated with 5 c.c. of concentrated ammonia. 
After stirring a little the solutions are set aside until the next day. 
The supernatant fluid is then carefully poured off through a filter 
(Schleicher and Schull, No. 597); the precipitated ammonium urate 



426 THE URINE 

is collected with the aid of a small amount of a 10 per cent, solution 
of ammonium sulphate and washed with the same reagent. Traces 
of chlorides do not interfere with the subsequent titration, and the 
process of filtration and washing can be completed in from twenty to 
thirty minutes. The ammonium urate is washed into a beaker, after 
opening the filter, using about 100 c.c. of water; 15 c.c. of con- 
centrated sulphuric acid are then added, and the solution is titrated 
at once with a one-twentieth normal solution of potassium permanga- 
nate. Toward the end of the titration Folin suggests to add the per- 
manganate in portions of two drops at a time, until the first trace of a 
rose color is apparent throughout the entire fluid. Each cubic centi- 
meter of the reagent corresponds to 0.00375 gram of uric acid. A 
final correction (of 0.003 gram for every 100 c.c. of urine employed) 
is necessary, owing to the slight extent to which ammonium urate is 
soluble. 

Preparation of the One-twentieth Normal Solution of Potassium 
Permanganate. — As the molecular weight of potassium permanga- 
nate is 157.67, one would expect that a normal solution of the salt 
should contain this amount in grams dissolved in 1000 c.c. of water. 
But the substance generally acts in the presence of free acids, upon 
deoxidizing substances, by losing 5 atoms of oxygen of the 8 atoms 
contained in 2 molecules, as is seen in the following equation: 

2KMn0 4 + 5H 2 C,0 4 + 3H 2 S0 4 = K 2 S0 4 + 2MnS0 4 + 10CO 2 + 8H 2 0. 

It follows that two-fifths of the molecular weight, or 63.068 grams, 
are the equivalent of 1 oxygen atom. But as oxygen is diatomic 
and the volumetric normal is calculated for monatomic values, this 
number must be divided by 2, and 31.534 grams of potassium per- 
manganate should therefore be present in 1 liter of normal solution. 
A one-tenth normal solution would hence contain 3.1534 grams, and 
a one-twentieth normal solution 1.576 grams pro liter. This amount is 
weighed off and dissolved in 950 c.c. of water, when the solution is 
brought to the proper degree of dilution by titration with a one- 
twentieth normal solution of oxalic acid. A one-twentieth normal 
solution of oxalic acid contains 3.142 grams of the acid in 1000 
c.c. of water. One c.c. of the one-twentieth normal solution of 
potassium permanganate should correspond to 1 c.c. of the oxalic 
acid solution. The titration is best conducted by diluting 10 c.c. 
of the oxalic acid solution to 100 c.c. with distilled water and add- 
ing 15 c.c. of concentrated sulphuric acid, so as to bring the tempera- 
ture of the liquid to from 55° to 65° C. The potassium perman- 
ganate solution is then added drop by drop until the red color no 
longer disappears on stirring, but persists for at least thirty seconds. 

For Salkowski's method of estimating uric acid see method for 
estimating the xanthin bases. 



CHEMISTRY OF THE URINE 427 

The Xanthin Bases. 

The xanthin bases which have been found in the urine are xanthin, 
hypoxanthin, heteroxanthin, paraxanthin, guanin, and adenin. Con- 
jointly they are also spoken of as the alloxur bases, or purin bases. 
Together with uric acid they are termed alloxur or purin bodies. 
Their relation to uric acid and the nucleins has already been con- 
sidered. (See Uric Acid.) Unlike uric acid, they also occur as such 
in animal as well as vegetable tissues. The amount which appears 
in the urine under normal conditions is very small, constituting 
about 10 per cent, of the uric acid. Larger quantities may be met 
with in various diseases, and, generally speaking, an increase in the 
amount of uric acid is associated with an increase of the xanthin 
bases. This is, however, not invariably the case, and at times it 
may be observed that an increase of the uric acid is accompanied by 
a diminution of the xanthins, and vice versa. These varying rela- 
tions can, of course, be readily understood if we remember that uric 
acid is an oxidation product of the xanthin bases, and that their 
ultimate origin is the same. ' The largest quantities of xanthin bases are 
found in leukemia; Magnus-Levy has reported a case with 0.321 gram. 

Individually the xanthin bases are of little clinical interest. 
Xanthin has once been found in a urinary sediment, and has in 
several instances been encountered as the principal constituent of 
vesical calculi. Its normal quantity is said to vary between 0.02 
and 0.03 gram. Larger quantities are found after a meal rich in 
nucleins, in leukemia, nephritis, pneumonia, etc. 

Paraxanthin and heteroxanthin are present only in traces, as is 
apparent from the fact that Kruger and Salomon were able to obtain 
but 7.5 grams of heteroxanthin from 10,000 liters of urine. Both 
apparently are distinctly toxic. 

Xanthin sediments may be recognized by means of the following 
test: A small amount of the material is treated with a few drops of 
concentrated nitric acid on a porcelain plate, and evaporated to dry- 
ness. In the presence of xanthin a yellow residue is obtained, which 
turns a violet red upon the addition of a few drops of sodium hydrate 
solution and the application of heat (Strecker's test). The reaction 
is common to all the xanthins and should not be confused with the 
murexid test. 

Quantitative Estimation. Salkowski's Method. 1 — 600 c.c. of urine 
are precipitated with 200 c.c. of magnesia mixture (composed of 
1 part of crystallized magnesium sulphate, 2 parts of ammonium 
chloride, 4 parts of ammonium hydrate, and 8 parts of distilled water) , 
when a 3 per cent, ammoniacal solution of silver nitrate is added to 
from 700 to 750 c.c. of the filtrate. The proportion should be 6 c.c. 

1 Pfliiger's Archiv, vol. lxix, p. 268. 



428 THE URINE 

for each 100 c.c. of urine. If the precipitated silver chloride formed 
in the beginning does not disappear on stirring, a little more ammo- 
nium hydrate is added. A flaky precipitate next separates out, and 
is allowed to settle. In order to tost whether enough of the silver 
nitrate solution has been added, a few cubic centimeters of the super- 
natant fluid are acidified with nitric acid. If a distinct cloudiness, 
referable to silver chloride, appears, enough has been added. Other- 
wise the few cubic centimeters that were employed for this test are 
rendered alkaline again with ammonia, poured back, and treated 
with more silver solution until the required amount has been reached. 
After standing for one hour the mixture is filtered and the precipitate 
washed with water until all the free silver has been removed. The 
filter is then perforated, the precipitate washed into a flask with 
from 600 to 800 c.c. of water, acidified with hydrochloric acid, and 
decomposed with hydrogen sulphide. The excess of hydrogen sul- 
phide is removed by heating on a water bath, when the silver sulphide 
is filtered off and the filtrate evaporated to dryness. The residue is 
treated with from 25 to 30 c.c. of dilute sulphuric acid (1 to 100). This 
solution is brought to the boiling point and is allowed to stand over 
night. The uric acid which has then separated out is filtered off, 
washed with a small amount of dilute sulphuric acid (not more than 
50 c.c), then with alcohol and ether, and weighed. To the resulting 
weight 0.0005 gram is added for each 10 c.c. of the acid filtrate, to 
allow for the trace of uric acid which is thus lost. 

After having filtered off the uric acid the filtrate is again treated 
with ammonia and silver solution, and the xanthin bases thus pre- 
cipitated. The precipitate is collected on a small filter, washed with 
water, dried, and incinerated. The ash is dissolved in nitric acid, 
and the silver estimated by titration with a solution of potassium 
sulphocyanide, using ammonioferric alum as an indicator. (See Chlo- 
rides.) The solution of potassium sulphocyanide employed in the 
estimation of the chlorides may be used, and is of such strength 
that 1 c.c. corresponds to 0.00734 gram of silver. As 1 atom of 
silver in a mixture of the silver compounds of guanin, xanthin, 
hypoxantin, etc., represents 0.277 gram of nitrogen, or 0.7381 gram 
of the alloxur bases, it is apparent that 1 c.c. of the potassium sul- 
phocyanide solution will represent 0.002 gram of nitrogen and 
0.00542 gram of alloxur bases. In every case an accurate record 
must, of course, be kept of the amount of urine and filtrate used. 

The amount of alloxur bases found by Salkowski in the normal 
urine of twenty-four hours varied between 0.0286 and 0.0561 gram. 

Literature. — M. Kruger u. G. Salomon, "Die Alloxurbasen d. Hams," Zeit. 
f. physiol. Chem., vol. xxiv, p. 364, and vol. xxvi, 343; Deutsch. med. Woch., 
1899, p. 97. Bondsynski u. Gottlieb. "Ueber Xanthinkorper im Harn des 
Leukamiker," Arch. f. exper. Path. u. Pharmakol., 1895, vol. xxxvi, p. 132 F. 
Gumprecht, "Alloxurkorper u. Leukocyten," Centralbl f. allg. Path. u. path. 
Anat., 1896, vol. vii, p. 820. 



CHEMISTRY OF THE URINE 429 

Hippuric Acid. 

Hippuric acid is a constant constituent of normal urine, 0.1 to 1 
gram being excreted in the twenty-four hours. That it is derived, 
to some extent at least, from albuminous material is proved by the 
fact that its elimination is not suspended during starvation nor during 
the administration of a purely albuminous diet. In vitro it may 
be obtained from glycocoll and benzoic acid, according to the equation 



p6 H 5 

1 + 

COOH 


CH 2 NH 2 

1 
COOH 


CH 2 NH— C 6 H 5 CO 
= 1 + H 2 0. 
COOH 


nzoic acid. 


Glycocoll. 


' Hippuric acid. 



It has been shown that phenylpropionic acid, which differs from 
benzoic acid by the group C 2 H 4 , and which may be regarded as 
phenylformic acid, is produced during the process of intestinal 
putrefaction. The relation between the two bodies is seen from the 
formulas : 

H C 6 H 5 CH 3 CH 2 .C 6 H 5 

I > I I I 

COOH COOH CH 2 > CH 2 

Formic Phenvlformic 

acid. acid. C00H C00H 

Propionic Phenylpropionic 
acid. acid. 

Phenylpropionic acid is then absorbed into the blood and there, 
according to our present ideas, transformed into phenylformic acid 
or benzoic acid. When the latter comes in contact with glycocoll, 
which is produced during the process of pancreatic digestion, an 
interaction between the two substances occurs in the body, hippuric 
acid resulting, as shown in the above equation. This view is sup- 
ported by the fact that phenylpropionic acid, just as benzoic acid, 
when introduced into the circulation of certain animals, reappears in 
the urine as hippuric acid. The final proof of the possible synthesis 
of hippuric acid from glycocoll and benzoic acid in the body has been 
furnished by Bunge and Schmiedeberg, 1 who obtained this substance, 
when arteriaii zed blood containing glycocoll and sodium benzoate 
was allowed to pass through isolated kidneys of dogs. 

Not all the hippuric acid eliminated, however, is referable to albu- 
minous decomposition, but a considerable portion is derived from 
benzoic acid or its derivatives, which occur in many fruits, and 
are transformed into hippuric acid in the body. Among those 
which are particularly rich in these substances may be mentioned 
the red bilberry, prunes, coffee-beans, green gages, etc., and in all 
cases in which an increased elimination of hippuric acid is observed 
the possibility of this source must be taken into account. 

1 Arch. f. exper, Path, u. Pharmakol., vol vi. 



430 



THE URINE 



As to the seat of the synthesis there appears to be some uncer- 
tainty, as it is apparently not the same in all animals. In the dog 
and the frog the kidneys, according to the researches of Bunge and 
Schmiedeberg, must be regarded as the principal and possibly the 
only organs in which this process occurs. As Salomon, however, 
has demonstrated the presence of hippuric acid in the muscles, liver, 
and blood of nephrectomized rabbits, still other organs must, in the 
herbivora at least, be concerned in its production. 

Very little is known of the pathological variations in the excre- 
tion of hippuric acid; this is principally owing to the fact that until 
recently suitable methods for its quantitative estimation were not 
available. It is an interesting fact that, in accordance with Bunge's 
experiments in dogs, the formation of hippuric acid appears to be 
suspended in cases of acute as well as chronic parenchymatous 
nephritis, for the benzoic acid which is then ingested reappears 




Fig. 144. — Hippuric acid crystals. 

in the urine unchanged. In amyloid degeneration a marked dimi- 
nution has likewise been demonstrated. Large quantities of hippuric 
acid, on the other hand, have been noted in acute febrile diseases, 
hepatic diseases, diabetes mellitus, chorea, etc. The data, how- 
ever, are insufficient to warrant any definite conclusions. 1 

Properties of Hippuric Acid. — Hippuric acid crystallizes in long, 
rhombic prisms when allowed to separate from its solutions gradually, 
while it forms long needles if crystallization takes place rapidly and 
the amount is small (Fig. 144). In cold water and ether it is soluble 
with difficulty, while it dissolves readily in hot water, in alcohol, and 
in aqueous solutions of the hydrates and carbonates of the alkalies, 
with which it forms salts, and from which the acid may again be 
separated and caused to crystallize out by adding a stronger acid. 

1 Th. Weyl u. B. von Anerep, "Ueber die Ausscheidung der Hippursaure und 
Benzoesaure wahrend des Fiebers," Zeit. f. physiol. Chem., 1880, vol. iv, p. 169 



CHEMISTRY OF THE URINE 431 

When hippuric acid or one of its salts is evaporated to dryness 
with concentrated nitric acid and the residue is heated, the odor of 
bitter almonds is noticed; this is due to the formation of nitrobenzol. 

When boiled with hydrochloric acid or dilute sulphuric acid 
hippuric acid is decomposed into glycocoll and benzoic acid. A 
similar decomposition is effected during the process of putrefaction, 
and hence no hippuric acid is found in decomposing urine, benzoic 
acid taking its place. The latter is always found in the urine together 
with hippuric acid, but has no clinical significance. In larger amounts 
it has been encountered in diabetes. It crystallizes in needles or 
lustrous lamina?, presenting ragged edges, which resemble plates of 
cholesterin. It is soluble with difficulty in cold water, but easily 
soluble in ether, alcohol, and solutions of the alkaline carbonates and 
hydrates, forming salts with the latter. 

Hippuric acid in the urine occurs in combination with sodium, 
potassium, calcium, and magnesium. 

Quantitative Estimation of Hippuric Acid. — The following 
method may be employed for the quantitative estimation of hippuric 
acid: 

Principle. — Hippuric acid readily dissolves in solutions of the 
alkaline hydrates and carbonates, forming salts. These are decom- 
posed by means of a stronger acid, when the hippuric acid which 
separates out is collected and weighed. 

Method. — 500 to 1000 c.c. of fresh urine are evaporated to a 
syrupy consistence on a water bath, care being taken to keep the 
urine neutral by the addition of sodium carbonate. The residue is 
extracted with cold alcohol (90 to 95 per cent.), using about half 
of the quantity as that of the urine employed. The mixture is 
then set aside for twenty-four hours. The alcoholic filtrate, which 
contains the salts of hippuric acid, is freed from alcohol by dis- 
tillation. The remaining solution is strongly acidified with acetic 
acid and extracted with at least five times its volume of alcoholic 
ether (1 part of alcohol to 9 parts of ether). From the combined 
extracts the ether is distilled off and the remaining solution evapo- 
rated on a water bath. The resinous residue is boiled with water, 
set aside to cool, and filtered through a well-moistened filter. The 
hippuric acid, which is easily soluble in boiling water, is thus 
separated from other constituents which are soluble in alcohol 
and ether. The filtrate is rendered alkaline with a little milk 
of lime, any excess of calcium being removed by passing carbon 
dioxide through the mixture. This is then brought to the boiling 
point and filtered. Any impurities which may be present are re- 
moved by shaking with ether. The calcium salts remaining in solu- 
tion are decomposed by means of an acid, when the solution is again 
extracted with ether. The remaining solution is evaporated to a few 
pubic centimeters, when the hippuric acid will separate out on stand- 



432 THE URINE 

ing. The crystals are dried on plates of plaster of Paris, shaken 
with benzol or petroleum ether to remove any benzoic acid, and 
finally weighed. These crystals may be shown to be hippuric acid 
by their microscopic appearance, their solubility in alcohol, and 
their behavior when evaporated with concentrated nitric acid, as 
indicated above. 

Hofmeister's Method. — 200 to 300 c.c. of urine are evaporated in 
a glass dish to one-third of the original volume, and treated with 
4 grams of disodium phosphate, to transform the acid into its sodium 
salt. The mixture is evaporated to a syrupy consistence, the resi- 
due treated with burnt gypsum, dried thoroughly, and pulverized 
together with the dish. The powder is extracted in a Soxhlet 
apparatus with freshly rectified petroleum ether (boiling point 60° 
to 80° C.) for forty-six hours, and then for six to ten hours with pure 
ether (free from water and alcohol). After distilling off the ether 
the residue is dissolved in boiling water and decolorized with animal 
charcoal, the latter being subsequently thoroughly washed with boil- 
ing water; the solution and washings are evaporated to about 1 or 2 
c.c. at a temperature of from 50° to 60° C, and set aside to crystallize. 
The crystals of hippuric acid are finally washed with a few drops 
of water and ether, and weighed. 



Ereatin and Kreatinin. 

The antecedents of kreatin and kreatinin are unknown. Two 
sources of the urinary kreatinin must be recognized, viz., the muscle 
tissue of the body and the muscle tissue ingested as food. The tissue 
kreatin is possibly transformed into kreatinin and eliminated in this 
form, while the kreatin which has been ingested does not appear in the 
urine as kreatinin. Its fate is not known. Folin regards kreatinin 
as the essential end product of the endogenous nitrogenous katab- 
olism, in so far at least as the muscle tissue is concerned, He 
has demonstrated the interesting fact that its absolute quantity on 
a meat-free diet is a constant quantity, which is different for different 
individuals, but wholly independent of quantitative changes in the total 
amount of nitrogen eliminated. Its relative amount is increased when 
the urea nitrogen falls. On a diet rich in proteids the kreatinin nitrogen 
represents 3.2 to 4.5 per cent, of the total, while on one free from pro- 
teids (starch and cream) the amount may rise to 17.4 per cent. The 
absolute amount seems to depend to a certain extent upon the body 
weight. Fat or corpulent persons yield less kreatinin per unit of body 
weight, namely, 20 mgrms. per kilo, while lean persons yield about 
25 mgrms. 1.15 to 1.6 grams may thus be regarded as average values. 

The study of pathological variations in the amount of kreatinin has 
been greatly facilitated through the introduction of Folin 's method 



CHEMISTRY OF THE URINE 433 

(see below). The older data are of little importance, unless the diet 
of the individual has been carefully considered. A diet rich in meats, 
it should be borne in mind, greatly increases the amount. 

If then in patients affected with acute febrile diseases, such as 
pneumonia, typhoid fever, etc., a large increase is observed, the 
patient being at the same time upon a milk diet, an increased 
destruction of muscle tissue may be inferred, as a milk diet in itself, 
cceteris paribus, causes a diminished elimination. A decrease would 
logically be expected to occur during convalescence from such dis- 
eases. In the various forms of anemia, marasmus, chlorosis, phthisis, 
etc., a diminished amount is observed. 1 The same is seen in advanced 
cases of chronic parenchymatous nephritis, in progressive muscular 
atrophy, in pseudohypertrophic paralysis, and in progressive ossifying 
myositis. 

Properties of Kreatin and Kreatinin. — Chemically, kreatin may 
be regarded as a methyl derivative of glucocyamin, which latter is 
guanidin in which 1 NH 2 group has been replaced by glycocoll. 
Kreatinin, on the other hand, is the methyl derivative of glucocy- 
amidin, which differs from glucocyamin only in the absence of 1 
molecule of water, so that kreatinin is kreatin minus 1 molecule of 
water, both being thus theoretically derivatives of guanidin. The 
relation between the various bodies is shown below: 

/NH 2 

C=NH 

\NH 2 

Guanidin. 

/NH 2 /NH 2 

C^NH C=NH 

\ NH.CH 2 .COOH \ N(CH 3 ) .CH 2 .COOH 

Glucocyamin. Kreatin. 

/NH /NH 

C=NH C=N 

\NH.CH 2 .CO \N(CH 3 ).CH 2 .CO 

Glucocyamidin (glucocyamin minus water). Kreatinin (kreatin minus water). 

Kreatinin crystallizes without water of crystallization in colorless, 
glistening prisms. At times, when the crystals are not well devel- 
oped, it also appears in the form of whetstones. It is readily soluble 
in hot and also quite soluble in cold water and hot alcohol; in cold 
alcohol and ether it dissolves with difficulty. It forms salts with 
acids, and double salts with some of the salts of the heavy metals. 
Among these may be mentioned kreatinin hydrochloride, C 4 H 7 N s O.- 
HC1, which is easily soluble in water and crystallizes in the form of 
transparent prisms or rhombic plates. Most important is the com- 
pound of kreatinin with zinc chloride, (C 4 H 7 N 3 0) 2 .ZnCl 2 (Fig. 145). 
This is produced when a watery or alcoholic solution of kreatinin is 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1907. Senator, 
Virchow's Archiv, 1876, vol. lxvii, p. 422. Neubauer u. Vogel, Harnanalyse, 
pt. ii. 

28 



434 



THE URINE 



treated with zinc chloride. The crystalline form of this compound 
depends greatly upon the purity of the kreatinin solution. When 
obtained from alcoholic extracts of the urine it occurs in the form 
of varicose conglomerations which often adhere firmly to the walls 
of the vessel. If the solution of kreatinin is perfectly pure, how- 
ever, it is seen in the form of fine needles grouped in rosettes or 
sheaves. Kreatinin-zinc chloride is soluble with much difficulty in 
water and insoluble in alcohol. The compound is especially impor- 
tant, as upon its formation and properties the quantitative estimation 
of kreatinin in the urine is based. Silver nitrate and mercuric chlo- 
ride cause a precipitation of kreatinin, and may, therefore, also be 
employed for the purpose of obtaining the substance from the urine. 




Fig. 145. — Crystals of kreatinin-zinc chloride. (Salkowski.) 



Test for Kreatinin in the Urine. — A few cubic centimeters of urine 
are treated with a few drops of a very dilute solution of sodium 
nitroprusside and then drop by drop with a dilute solution of sodium 
hydrate. In the presence of kreatinin the urine assumes a ruby-red 
color, which is particularly well seen in the lower portion of the 
tube. This color disappears after a few minutes, and is replaced by 
an intense yellow, which on warming with glacial acetic acid in pure 
solutions turns to green, then to blue, and on standing a deposit of 
Prussian blue is obtained (WeyVs test). 1 The presence of albumin 
or sugar does not interfere with the reaction. 

Folin's Method. 2 — This method is based on Jaffe's reaction of 
kreatinin with alkaline picric acid solution. The red-colored solu- 
tion produced in this reaction has in proper concentration and when 
viewed by transmitted light exactly the same shade as a potassium 

1 Th. Weyl, Ber. d. deutsch. chem. Gesellsch., 1878, vol. xi, p. 217; and Jaffe, 
Zeit. f. physiol. Chem., 1886, vol. x, p. 399. 

? The above description of the method I gwe to the courtesy of Dr. Folin, 



CHEMISTRY OF THE URINE 435 

bichromate solution. Half-normal potassium bichromate solution 
(containing 24.55 grams per liter) is therefore used as a standard 
for comparison. A high-grade colorimeter, by means of which the 
depths both of the unknown solution and of the bichromate can be 
adjusted to tenths of millimeters, is necessary for the comparison. 1 

The following solutions are also necessary: The half -normal potas- 
sium bichromate solution, 10 per cent, sodic hydrate, and a satu- 
rated (1.2 per cent.) picric acid solution. 

If to 10 mgrms. of chemically pure kreatinin dissolved in 10 c.c. 
of water in a 500 c.c. volumetric flask are added 15 c.c. of picric 
acid solution and 5 c.c. of sodic hydrate, the maximum color is ob- 
tained at the end of five minutes. If at the end of this time the 
solution be diluted to the 500 c.c. mark and at once compared with 
the standard bichromate solution, it will be found that 8.1 mm. of 
the kreatinin-picrate solution have in the colorimeter exactly the 
same shade and depth of color as 8 mm. of the bichromate solution. 

The actual determination in urine is carried out in exactly the 

same way, substituting 10 c.c. of urine for the kreatinin solution. The 

more kreatinin that is present in the 10 c.c. of urine the deeper will, 

of course, be the color of the solution obtained. Supposing the 

colorimetric observation shows that 7.1 mm. of the urine-picrate 

solution are equal in color to 8 mm. of the standard, the 10 c.c. 

8 7 
of urine would then contain 10 X ^'- = 11.4 mgrms. of kreatinin. 

The following precautions are to be observed in the determination : 

1. Make first a preliminary colorimetric observation, using half- 
normal potassium bichromate solution in both cylinders of the 
colorimeter, adjusting first one to the 8 mm. mark. The average of 
three or four readings of the other cylinder should also be 8 mm., 
and after the first observation no two should differ by more than 0.2 
mm. This preliminary observation takes only two or three minutes, 
and is exceedingly useful in making the eye sure of the correct point 
to be ascertained. 

2. Exactly 8 mm. of the half-normal potassium bichromate solu- 
tion must be used as the standard for comparison. 16 or 24 mm., 
for example, cannot be substituted on the basis of the calculation 
given above because the kreatinin-picrate solution absorbs light at 
an entirely different rate from that of the bichromate solution. 

3. For the reason given in the preceding paragraph it is necessary 
to make each determination with a quantity of urine containing not 
less than 5 nor more than 15 mgrms. of kreatinin. Within these 
limits the determination as described is correct within 0.2 mgrm. 

4. Sugar and albumin do not interfere with the determination. 
Acetone, diacetic acid, and hydrogen sulphide do interfere. Where 

1 The French instrument of Duboscq, which can be obtained through Eimer & 
Amend, is admirably suited for the purpose. 



436 THE URINE 

these are present the urine should be measured into a porcelain evap- 
orating dish and heated on a water bath with 10 c.c. of 1 per cent, 
hydrochloric acid for about half an hour. When the dish is again 
cooled, the reagents are added directly into the dish, and finally 
rinsed into the volumetric flask after five minutes. 

5. The color due to the urine is ordinarily of no appreciable con- 
sequence because of the great dilution. Urines containing bile pig- 
ments can, however, first be cleared by the addition of egg albumen 
and then removing this by coagulation (heat). 

The whole operation can be finished in less than fifteen minutes; 
indeed, it should be finished at once, as the colored product obtained 
by the interaction of kreatinin and picric acid is not very stable. 



Oxalic Acid. 

The origin of oxalic acid in normal urine is twofold. The greater 
portion is supposedly derived from the ingested food, but there 
is evidence to show that a certain amount is also formed during 
the metabolism of the body tissues, as the elimination of oxalic acid 
does not cease during starvation. The carbohydrates and fats 
probably do not play a part in this connection; and, according to 
Salkowski, the albumins also do not enter into consideration per se. 
He rather inclines to the view that the nucleins represent the 
antecedent of the oxalic acid, and as a matter of fact uric acid, which, 
as we have seen, is itself derived from the nucleinic bases, can be 
readily oxidized to oxalic acid, with the intermediary formation of 
parabamic acid and oxaluric acid. The latter has been repeatedly 
demonstrated in the urine, and it is conceivable that the same pro- 
cess may occur in the animal body. But even supposing that the 
oxaluric acid which is obtained from the urine is formed artificially 
during the lengthy process of analysis, and that the substance did 
not exist preformed, there is no reason for the assumption that uric 
acid may not be the normal antecedent of the oxalic acid. For 
Salkowski has demonstrated conclusively that on oxidation with 
ferric chloride in aqueous solution uric acid yields oxalic acid and 
urea directly. 

The matter, however, is not quite so simple as it appears, and an 
increased elimination of oxalic acid by no means always occurs 
when the output of uric acid is increased. After the ingestion of 
fairly large amounts of thymus, for example, the usual increase of 
uric acid is not accompanied by a corresponding increase in the 
amount of oxalic acid, and in those cases in which it does occur 
we are as yet unable to exclude the large amount of connective 
tissue as the source of the oxalic acid. Connective tissue and gelatin 
have, as a matter of fact, been shown to increase the amount of 



CHEMISTRY OF THE URINE 437 

oxalic acid when given in large amounts. With pure nuclein no 
effect has been observed, and it can be shown that in those experi- 
ments in which this was used by mouth an absorption from the 
intestinal tract had manifestly not occurred (Mohr and Salomon). 1 

Under pathological conditions oxalic acid may also be formed 
in the digestive tract from the ingested carbohydrates, as a result 
of a peculiar fermentative process. This has been well shown 
by Helen Baldwin in Herter's laboratory. In some of these cases 
no free hydrochloric acid could be demonstrated in the gastric con- 
tents, and it was observed that inoculation of a digestive mixture, 
which was originally free from oxalic acid, resulted in its appearance 
if a few drops of such stomach contents were added. In dogs pro- 
longed feeding with excessive quantities of glucose together with 
meat was seen to lead eventually to a state of oxaluria, which was 
associated with a mucous gastritis and the absence of free hydro- 
chloric acid. Oxalic acid could then also be demonstrated in the 
stomach contents. 

Very curiously the ingestion of quite small and non-toxic amounts 
of oxalic acid is followed . by a fairly intense indicanuria. It does 
not seem likely to me, however, that as Harnack and v. d. Leyen 
suggest, the indicanuria is here referable to a toxic action upon the 
tissue albumins, and I am personally inclined to explain the phe- 
nomenon upon the basis of increased intestinal putrefaction. (See 
Indicanuria.) 

The amount of oxalic acid which is normally eliminated in the 
twenty-four hours fluctuates with the amount ingested, and varies 
from a few milligrams to 2 or 3 centigrams, being usually less 
than 10 milligrams (Baldwin). It is influenced by the character 
of the diet. The ingestion of oxalates by the mouth is followed 
by their partial elimination only in urine and feces, so that we may 
conclude that to a certain extent oxalic acid is decomposed during 
its passage through the animal body; possibly this may occur in the 
intestinal canal as the result of bacterial action. 

Foods rich in oxalic acid are spinach, tomatoes, carrots, celery, 
string-beans, rhubarb, potato, dried figs, plums, strawberries, cocoa, 
tea, coffee, and pepper. Foods which contain little or no oxalic 
acid, on the other hand, are meat, milk, eggs, butter, cornmeal, rice, 
peas, asparagus, cucumbers, mushrooms, onions, lettuce, cauliflower, 
pears, peaches, grapes, melons, and wheat, rye, and oat flour. 

Before drawing conclusions as to the existence of abnormal oxaluria 
it is hence imperative to eliminate the possibility of an increased 
ingestion, by placing the patient upon a diet which contains little 
or no oxalic acid. 

1 Deutsch. Arch. f. klin. Med;, 1901, vol. lxx, p. 486. Lommel, ibid., vol. lxiii, 
p 599 



438 THE URINE 

An increased elimination is notably observed in association with 
various dyspeptic and nervous manifestations, and constitutes the 
condition commonly spoken of as the oxalic acid diathesis, or as 
idiopathic oxaluria. Its existence as a definite pathological picture 
is, however, denied by most modern clinicians. Nevertheless it 
must be admitted that there is a certain type of neurasthenia in 
which, generally in association with hyperchlorhydria, an increased 
elimination of oxalic acid takes place, and in which a copious deposit 
of calcium oxalate crystals is frequently observed. From the mere 
fact of the occurrence of such deposits, of course, no inference is, 
as a rule, to be drawn regarding the actual elimination, but its fre- 
quent occurrence is in itself of importance, as in such cases a similar 
separation from the urine may already occur within the urinary 
passages, and not uncommonly in the pelvis of the kidneys. Not 
infrequently oxaluria of this type is associated with an increased 
elimination of uric acid and a mild grade of albuminuria, as has 
been shown by Senator, von Noorden, Da Costa, myself, and others. 
Whether or not the oxaluria in these cases can be explained upon 
the basis of abnormal fermentations in the gastro-intestinal tract, 
as is suggested by the observations of Baldwin, remains to be seen. 
In some this may be the case, but in others I am inclined to asso- 
ciate the oxaluria with the coexistent lithuria. 

Very interesting is the apparently vicarious oxaluria which is at times 
observed in diabetes. Fiirbringer has reported a case of diabetes in 
which the elimination of oxalic acid was described as " enormous, " 
and in which oxalic acid could also be demonstrated in the sputum 
(oxaloptysis). Rausch has recorded a case of mild diabetes, associated 
with hepatic cirrhosis, in which 1.2 grams were excreted in twenty- 
four hours. In most cases of diabetes, on the other hand, an in- 
creased oxaluria cannot be demonstrated. 

In cases of obesity Kisch found no abnormal degree of oxaluria. 

In association with jaundice increased oxaluria has been repeatedly 
observed, and is probably referable to biliary stasis and consequent 
cholemia, as Salkowski has demonstrated that the bile contains 
oxalic acid. In pneumonia and leukemia, in both of which we find 
as a rule a greatly increased elimination of uric acid, the oxalic acid 
is not always increased, and sometimes indeed quite low in comparison 
with the amount of uric acid. 

Properties of Oxalic Acid. — Oxalic acid occurs in the urine as cal- 
cium oxalate, CaC 2 4 , and is held in solution by the diacid sodium 
phosphate. It can, hence, be precipitated by diminishing the acidity 
of the urine by the addition of a little ammonia or by allowing it 
to stand exposed to the air. When allowed to crystallize out slowly, 
calcium oxalate occurs in the form of well-defined, strongly refract- 
ive octahedra, in which the principal axis of the crystals is placed 
at right angles to the plane of the microscopic slide (Fig. 146). These 



CHEMISTR Y OF THE URINE 



439 



are very characteristic. Other forms, however, are also quite com- 
monly observed, such as single and double dumb-bells, spheroids 
and prisms, etc. They are insoluble in ammonia and alcohol, almost 
insoluble in hot and cold water, and very slightly soluble in acetic 
acid, but dissolve with ease in the mineral acids. 

When strongly heated, the salt is decomposed into calcium oxide, 
carbon dioxide, and carbon monoxide. 

Tests for Oxalic Acid. — For the detection of calcium oxalate it is 
frequently only necessary to examine the sediment of the urine after 
standing for twenty-four to forty-eight hours. No oxalate crystals, 
however, may be found even when an abnormally large amount 
can be demonstrated by chemical methods. In such cases it is 
usually possible to bring about the crystallization of the salt by 
carefully neutralizing the urine with a little ammonia. Should this 
procedure not lead to the desired end, it is best to treat the urine 
with one-third its volume of 95 per cent, alcohol. The mixture is 
set aside for twenty-four to forty-eight 
hours, when the sediment is centrifugal- 
ized and examined with the microscope. 
This method, Baldwin states, represents 
a more delicate test for oxalic acid than 
the complicated methods of quantitative 
analysis which are available. 

Quantitative Estimation. — Heretofore 
the old method of Neubauer has been in 
general use, but it is at best unsatisfactory. 
It has been replaced by the methods of 
Dunlop and Salkowski. 

Dunlop's Method (slightly modified by 
Baldwin) . — In this case the calcium oxalate 

is precipitated from an acid solution by means of alcohol, instead 
of from an alkaline solution by calcium chloride. The urine is 
thymolized, and, if alkaline, acidified with a trace of acetic acid; 
500 c.c. of a well-mixed specimen of the collected urine of twenty- 
four hours are treated with 150 c.c. of over 90 per cent, alcohol, 
to precipitate the calcium oxalate. The mixture is set aside for 
forty-eight hours. It is then filtered, care being taken to ensure 
the entire removal of the crystals from the beaker. The sediment 
is thoroughly washed with hot and cold water, and finally with 
dilute acetic acid (1 per cent, solution). The filter is placed in a 
small beaker and soaked in a small amount of dilute hydrochloric 
acid. It is then washed with hot water until the washings no longer 
give an acid reaction. The acid solution and washings are filtered, 
and the filtrate evaporated to about 20 c.c. This is treated with a 
very small amount of a solution of calcium chloride, to ensure the 
presence of an excess of calcium. The solution is neutralized with 




Fig. 146. — Calcium oxalate 
crystals. 



440 THE URINE 

ammonia, slightly acidified with acetic acid, and treated with strong 
alcohol, so that the mixture contains 50 per cent. After forty-eight 
hours the sediment is collected on a filter free from mineral ash, and 
is washed with cold water and dilute acetic acid until free from chlo- 
rides. The filter with its contents is then incinerated, first over a 
Bunsen burner, and afterward for five minutes in a blow-pipe flame. 
On cooling over sulphuric acid the ash is weighed; the result multi- 
plied by 1.6 represents the amount of oxalic acid in the volume of 
urine examined. 

Salkowski's Method. — In the case of human urine of moderate 
concentration 500 c.c. of the non-filtered urine are evaporated to 
about one-third. On cooling, the liquid is acidified with 20 c.c. of 
hydrochloric acid (sp. gr. 1.12), and extracted three times with new 
portions of 200 c.c. each of a mixture of 9 to 10 volumes of ether 
and 1 volume of alcohol. The ethereal extracts, which contain 
the liberated oxalic acid are carefully separated from the urine 
and filtered through a dry filter. The ether is distilled off; the re- 
maining alcoholic solution, which still contains a little ether, is placed 
in a deep evaporating dish, diluted with 10 to 15 c.c. of water, and 
evaporated on a water bath. The resulting milky fluid is concen- 
trated, more water being added if necessary, until it becomes clear 
and a gummy material separates out. On cooling, the liquid, which 
should measure about 20 c.c, is passed through a small filter. This 
is washed once or twice with a little water, when filtrate and washings 
are rendered slightly alkaline with ammonia, treated with 1 to 2 c.c. 
of a 10 per cent, solution of calcium chloride, and acidified with 
dilute acetic acid. The reaction should be distinctly acid, but an 
excess should be avoided. An indication that a sufficient amount 
has been added is afforded by the dissolution of the precipitate of 
phosphates, which occurs after the addition of the calcium chloride 
solution. After standing for twenty-four hours, or, still better, forty- 
eight hours, the calcium oxalate that has separated out is collected 
on a filter free from ash, washed with hot and cold water, dried, and 
incinerated as usual (see above). The resulting weight multiplied 
by 1.6 indicates the corresponding amount of oxalic acid in grams. 

Literature. — P. Fiirbringer, " Zur Oxalsaureausscheidung durch d. Harn," 
Deutsch. Arch. f. klin. Med./'1876, vol. xviii, p. 143. J. C. Dunlop, "The Elimi- 
nation of Oxalic Acid in the Urine," etc., Jour. Path, and Bact., 1896 (an histori- 
cal review of the subject of oxaluria is here also given). H. Baldwin," An Experi- 
mental Studv of Oxaluria," Jour. Exper. Med., vol. v, p. 27. E. Salkowski, 
Berlin, klin. Woch., 1900, p. 434; and Zeit. f. physiol. Chem., vol. xxix, p. 437. 
E. Harnack, " Ueber Indicanurie in Folge von Oxalsaurewirkung," Zeit. f. 
physiol. Chem., 1900, vol. xxix, p. 205. 

Albumins. 

The albumins which may be met with in the urine are serum 
albumin, serum globulin, albumoses (peptones), the albumin of 



CHEMISTRY OF THE URINE 441 

Bence Jones, hemoglobin, nucleo-albumin, fibrin, histon, and nucleo- 
histon. Of these, serum albumin is the most important from a 
clinical standpoint. 

Serum Albumin. — The question whether or not serum albumin 
occurs normally in the urine — ?'. e., under strictly physiological con- 
ditions — has been much disputed. It is claimed by some that traces 
may be temporarily met with in apparently healthy individuals after 
severe muscular exercise, cold baths, mental labor, severe emotions, 
during menstruation, digestion, etc. This so-called physiological albu- 
minuria mostly occurs in young adults, and is usually, if not always, 
of brief duration. The urine, it is claimed, is otherwise normal — 
i. e., of normal amount, appearance, specific gravity, and compo- 
sition, and free from abnormal morphological constituents, such as 
casts, red corpuscles, leukocytes, and epithelial cells. 1 However, 
Darling 2 has shown that severe muscular exercise may produce a 
urinary picture which, even though temporary, closely simulates 
what is seen in acute nephritis. He reports 0.9 per cent, of albumin 
in a member of a Harvard four-oared crew after a two-mile race, and 
amounts varying from 0.25 to 0.5 per cent, in five others under similar 
conditions. The sediment at the same time contained large numbers 
of hyaline and finely granular casts, many with renal cells and red 
blood corpuscles adherent. In many of the sediments there were 
also numerous red cells as such and an excess of leukocytes. 

The existence of a physiological albuminuria, on the other hand, 
is denied, and the occurrence of serum albumin at least regarded as 
pathological in every case. I have never been able to convince my- 
self of the occurrence of serum albumin in the urine under strictly 
physiological conditions, and am hardly prepared to regard severe 
muscular and mental labor, severe mental emotions, cold baths, etc., 
as physiological stimuli. The albuminuria, so often observed during the 
first days of life, at which time sediments of uric acid and urates, mucus, 
epithelial cells from the different portions of the urinary tract, and 
even casts may also be seen — i. e., constituents which in adults 
would rightly be regarded as abnormal — has also been brought for- 
ward in support of the theory of a physiological albuminuria. There 
can be no doubt, however, that this form of albuminuria is referable 
to the profound changes that take place in the circulatory system 
after birth, and to some extent perhaps also to the well-known uric 
acid infarctions so frequently seen in the kidneys of the newly born, 
so that it would probably be better and more in accord with the 
teachings of pathology to regard this form of albuminuria also as 
abnormal. 3 

The more closely the subject of the so-called physiological albu- 

1 C. E. Simon, "Functional .Albuminuria, " N. Y. Med. Jour., 1895, p. 330. 

2 Boston Med. and Surg. Jour., September 7, 1899, p. 231. 

3 L. Landi, L'albuminuria nel parto, Morgagni, 1890, vol. xxxii 



442 THE URINE 

minuria is studied, the more improbable does its physiological nature 
appear, and a more detailed study of the metabolic processes, it may 
be confidently asserted, will ultimately lead to the conclusion that 
the presence of albumin in every case is a pathological phenomenon. 

The association of an increased elimination of urea and uric acid 
with albuminuria in apparently healthy individuals was noted many 
years ago, but received comparatively little attention. 1 Personal obser- 
vations have led me to look upon this form of albuminuria as of 
common occurrence, and while in almost every case the albumin can 
be caused to disappear from the urine by proper diet and exercise, 
there can be no doubt that, if neglected, granular atrophy may ulti- 
mately result. 

An albuminuria may at times be observed in anemic children 
and adolescents, and particularly in masturbating boys of the mouth- 
breathing type, but can hardly be regarded as physiological. The 
same may be said of the albuminuria of pregnancy and parturition. 

As regards the action of cold baths, Rem-Picci 2 reports that albu- 
minuria may be considered a constant phenomenon after cold baths, 
but that different subjects react differently under the same con- 
ditions. Those which show albuminuria more readily are, as a 
rule, the less robust and thinner individuals, such as are most sensi- 
tive to cold. The limits of temperature necessary to produce the 
phenomenon are from 12° to 13° C, when the immersion is not 
longer than three minutes. If the temperature be from 15° to 20° C, 
the albumin appears only after fifteen minutes' immersion. Above 
this temperature albuminuria does not occur, even if the bath lasts 
much longer. The colder the bath, the more rapid the appearance 
of albumin. The degree of albuminuria is always slight, and even 
in the more marked cases rarely exceeds 0.25 pro mille. The sedi- 
ment, according to Rem-Picci, occasionally shows a few hyaline 
casts, and often crystals of calcium oxalate. 

The course which may be taken by these various forms of what 
should be termed functional albuminuria, in which the amount of 
albumin rarely exceeds 0.1 per cent., is very interesting. The elimi- 
nation of albumin may thus be quite transitory on the one hand, as 
when following severe muscular exercise, cold baths, and the like. 
It may, however, also last for several days, or even weeks, and be 
followed by a disappearance of the albumin for a variable length of 
time, and again by its reappearance and continuance for days and 
weeks. The term intermittent albuminuria? has been applied to this 
latter type. At times the albuminuria may follow a definite course, 

1 Da Costa, '" The Albuminuria and Bright's Disease of Uric Acid and Oxalic 
Acid," Amer. Jour. Med. Sci., 1895. 

2 "On Albuminuria after Cold Baths." II Policlinico, 1901, vol. viii, p. 389. 

3 Bull, Berlin, klin. Woch., 1886, vol. xxiii, p. 717. Mareau, Rev. de med., 1886, 
vol. vi, p. 855. Klemperer, Zeit. f. klin. Med., 1887, vol. xii, p. 168. 



CHEMISTRY OF THE URINE 443 

disappearing and reappearing with such regularity that it has not 
improperly been styled cyclic albuminuria. 1 In this form the albu- 
min generally disappears from the urine during the night or during 
prolonged rest in bed, and reappears during the day, the erect pos- 
ture apparently favoring its reappearance; the term postural or 
orthostatic albuminuria has hence also been suggested for this form. 
Oswald, who made a careful study of cyclic albuminuria in Riegel's 
clinic, regards its occurrence as distinctly pathological, and as indi- 
cating the existence of nephritis. Remembering the importance of 
the subject, it may not be out of place to enumerate the reasons 
which led Oswald 2 to this conclusion: 

1. The patients generally come to the physician complaining of 
certain definite symptoms which are similar to those noted in cases 
of true nephritis. At times, however, no complaints are made, be- 
cause the patients have reasons for concealing them (as in examina- 
tions for life insurance), or because they are temporarily absent. 

2. The subjective complaints, as well as the anemia so frequently 
observed in such cases, generally disappear, together with the albu- 
min, under suitable treatment, and reappear when the anemia again 
becomes marked. 

3. In many a history of an antecedent nephritis the result of 
scarlatina or diphtheria may be obtained, as in 3 cases of Heub- 
ner, in 14 cases out of 20 described by Johnson, etc. In some 
also a direct transition from an acute nephritis to the cyclic form 
of albuminuria has been noted. Where this was not possible the 
history of an acute infectious disease or an angina that had been 
overlooked in the clinical history must be regarded as a possible cause. 

4. The absence of morphological elements, especially tube casts, 
does not exclude a nephritis. A large number of cases, moreover, 
have recently been observed in which casts were repeatedly found. 

5. A cyclic albuminuria may be observed in many cases of chronic 
nephritis. 

6. Marked organic abnormalities (such as heart lesions) need not 
be demonstrable, as they may be absent for a long period of time or 
may be unrecognizable. 

According to the researches of Erlanger and Hooker 3 orthostatic 
albuminuria is dependent upon a lowering of the pulse pressure 
(being the difference between the minimum and the maximum blood 
pressure), which constantly occurs when the individual changes from 
the recumbent to the erect posture. In the true form of orthostatic 
albuminuria the albumin present is serum albumin. Casts are absent. 4 

1 A. Keller, Beitrage z. Kenntniss d. cyklischen Albuminuric, Diss., Breslau, 
1896. 

2 " Cyklische Albuminurie u. Nephritis," Zeit. f. klin. Med., vol. xxvii, p. 73. 

3 Johns Hopkins Hosp. Reports, 1904, p. 346. 

4 Teissier, Rev. de med., April 10, 1905, p. 233. 



444 THE URINE 

It may be safely asserted that a transitory, intermittent, and cyclic 
albuminuria is not infrequently observed in apparently healthy indi- 
viduals, but that the facts so far brought forward do not warrant the 
assumption that such forms of albuminuria are physiological. 1 The 
occurrence of such albuminuria unquestionably demonstrates a cer- 
tain insufficiency of the renal epithelium, and I am much in favor, 
as Martius has proposed, of discarding the term physiological 
albuminuria altogether, and to speak of these various forms col- 
lectively as constitutional albuminuria. 

It would lead too far to enter into a detailed consideration of the 
various causes that have from time to time been suggested as an ex- 
planation of the fact that albumin does not occur in the urine under 
normal conditions. There can be no doubt, however, that the integ- 
rity of the epithelial lining of the glomeruli and the convoluted 
tubules must be regarded as the principal factor which prevents the 
albumin of the blood from passing into the urine. When the readi- 
ness with which the glandular structures of the kidney respond to 
any abnormal stimulation is considered, it is easily understood how 
an albuminuria may be produced in many different ways. Aside 
from acute and chronic inflammatory processes in the widest sense 
of the word, an albuminuria may be the result of circulatory dis- 
turbances in the kidneys of whatever kind — i. e., the result of anemia 
as well as of hyperemia. In many and perhaps the majority of cases 
of what Bamberger 2 terms hematogenous albuminuria, we have direct 
evidence of the existence of circulatory disturbances, as in cases of 
uncompensated valvular lesion, weak heart, emphysema, hepatic 
cirrhosis, etc. In other cases, however, the existence of such dis- 
turbances can only be surmised, and the question, whether or not 
the albuminuria observed in the various infectious diseases> for ex- 
ample, is referable to circulatory abnormalities or to a direct irrita- 
tive action of microbic poisons upon the renal parenchyma, must 
still remain open. 

From personal studies in connection with the functional albu- 
minuria of Da Costa, it seems not unlikely that in many cases in 
which obscure circulatory disturbances are supposed to exist and are 
held responsible for an existing albuminuria, this is referable rather 
to the strain thrown upon the kidneys by the continued elimination 
of abnormally large quantities of organic material, the quantity of 
water being at the same time proportionately small. 

If it is remembered, furthermore, that injuries affecting certain 
portions of the brain are followed by albuminuria, and that this 
may be artificially produced by a piquure, analogous to the glucosuric 

1 v. Noorden, Deutsch. Arch. f. klin. Med., vol. xxxviii, pp. 3 and 205. Leube, 
Zeit. f. klin. Med., 1887, vol. xiii, p. 1. Winternitz, Zeit. f. physiol. Chem., 1891, 
vol. xv, p. 189. C. E. Simon, loc. cit. 

2 Wien. med. Woch., 1881, pp. 145 and 177. 



CHEMISTRY OF THE URINE 445 

piquure of C. Bernard, still another factor is given which may possibly 
enter into the causation of albuminuria. 

Obstruction to the outflow of urine from the kidneys has also 
been experimentally shown to lead to albuminuria, an observation 
with which clinical experience is in perfect accord. 

In patients actually in labor albuminuria is common, and sup- 
posedly due to increased blood pressure in the kidneys caused by 
uterine contractions and the general disturbance of the circulation. 
The relative frequency of its occurrence is a matter of dispute, 
however, and widely differing statements are made by different 
observers, ranging from 15 to 20 per cent. (Petit, Winckel) to 99 and 
100 per cent. (Trautenroth, Pajikull). 

As regards the occurrence of albuminuria in pregnancy the results 
of different observers likewise differ, viz., from 1 to 50 per cent. 
In the last months of pregnancy Zangemeister 1 found albumin in 10 
per cent, of the cases examined, and if repeated examinations were 
made positive results were obtained persistently during the last three 
months in 40 per cent. The albuminuria is supposedly referable to 
some metabolic disturbance and impaired excretion by the kidneys. 

Finally, an abnormal composition of the blood may at times cause 
the albuminuria. 

In passing on to a more detailed study of the various pathological 
conditions in which an elimination of albumin may be noted, an 
attempt will be made to classify the various forms of albuminuria 
in accordance with the more general considerations set forth above. 
It should be remembered, however, as already indicated, that it may 
be very difficult, if not impossible, to assign one single cause to a 
given clinical case, as several factors may at the same time be operative 
in the production of the albuminuria. 

1. Functional Albuminuria. — Under this heading may be 
comprised the various forms of " physiological" albuminuria, which 
have already been considered. 

2. The Albuminuria associated with Organic Diseases of 
the Kidneys, viz., acute and chronic nephritis, renal arteriosclerosis, 
amyloid degeneration of the kidneys. 2 

In acute nephritis, albuminuria, usually of great intensity, is a 
constant and most important symptom. The amount eliminated 
is generally proportionate to the intensity of the disease, but varies 
within fairly wide limits, generally from 0.3 to 1 per cent., corre- 
sponding to a daily excretion of from 5 to 8 grams. Much larger 
quantities, it is true, are at times excreted, but it may be definitely 
stated that the daily loss of albumin seldom exceeds 20 grams. 

In chronic parenchymatous nephritis the elimination of albumin 
is likewise constant, and the amount excreted in severe cases may 

1 Arch. f. Gyn., 1902, vol. lxvi, Heft 2. 

2 Senator, loc. cit. 



446 THE URINE 

even exceed that observed in the acute form. An elimination of 
from 15 to 30 grams, viz., 1.5 to 3 per cent, by weight, is frequently 
observed. 

In the ordinary form of chronic interstitial nephritis the elimina- 
tion of albumin is, as a general rule, slight, and rarely amounts to 
more than 2 to 5 grams pro die. At the same time it is not unusual 
to meet with an apparent absence of albumin if the more com- 
mon tests (see below) are employed. If it is remembered that 
very often the diagnosis of the disease is dependent upon the demon- 
stration of the presence or absence of albumin, the necessity of fre- 
quent examinations and the employment of more delicate tests, par- 
ticularly of the trichloracetic acid test, as well as of a microscopic 
examination, is at once apparent. This is even of greater impor- 
tance in the renal arteriosclerosis of Senator, in which albumin by 
the ordinary tests is probably not demonstrable in the majority of 
cases, and in which even the trichloracetic acid test may not be of 
service, and casts be absent. 

Amyloid degeneration of the kidneys, in the absence of inflamma- 
tory processes, is accompanied by a condition of the urine closely 
resembling that observed in the ordinary form of chronic interstitial 
nephritis. A total absence of albumin, however, is less frequently 
noted, while an amount varying between 1 and 2 per cent, is not 
uncommon. It will be shown later on that in this condition con- 
siderable amounts of serum globulin are excreted in addition to 
the serum albumin; larger amounts, in fact, than are generally 
observed in this form of chronic renal disease; so that Senator sug- 
gests that such a relation, in the absence of an acute nephritis, or 
an acute exacerbation of a chronic nephritis, may be of a certain 
diagnostic value. 

3. Febrile Albuminuria. 1 — That albuminuria may occur in 
almost any one of the various febrile diseases is a well-known fact, 
but it is important to remember that, while such an albuminuria 
may at times be referable to a true nephritis developing in the course 
of or during convalescence from an acute febrile disease, such is the 
exception, and not the rule. Under this heading, only that form 
will be considered which is not associated with distinct changes 
affecting the renal parenchyma, and which generally appears during 
the height of the disease only, and disappears with a return of the 
temperature to normal. As has been mentioned, it is often diffi- 
cult, if not impossible, to assign a definite cause for an albuminuria 
of this character, and in all probability several factors are in opera- 
tion at the same time. In the beginning of the disease, when the 
blood pressure, as a rule, is increased, the albuminuria may be 

1 Leyden, Zeit. f. klin. Med., 1881, vol. iii, p. 161. H. Lorenz, Wien. klin. 
Woch., 1888, vol. i, p, 119, 



CHEMISTRY OF THE URINE 447 

referable to an ischemia of the kidneys, as the increased pressure 
in fever, according to Cohnheim and Mendelson, is largely refer- 
able to spasm of the arterioles. Later on, or in the beginning of cases 
in which especially severe intoxication exists, the blood pressure may 
be subnormal, and the albuminuria be due to this cause — i. e., a hyper- 
emic condition of the kidneys. As a matter of fact, it has been experi- 
mentally demonstrated that both anemia and hyperemia of the kidney 
structure may lead to albuminuria. On the other hand, it is not 
unlikely that the strain thrown upon the kidneys by an excessive 
elimination of organic material, in the absence of a correspondingly 
large quantity of water, may produce albuminuria. I have Repeatedly 
seen the functional albuminuria of the type described by Da Costa 
disappear during the administration of a diet relatively poor in nitro- 
gen, while an increased diuresis was at the same time effected by the 
consumption of large amounts of water. 

In those grave cases of typhoid fever, furthermore, which are 
characterized by high fever and pronounced nervous symptoms, it 
would appear quite likely that the albuminuria, which in these cases 
is particularly marked, is referable to a direct influence upon the 
central nervous system, and in some cases, at least, also dependent 
upon an irritant action upon the renal epithelium on the part of the 
microbic poisons circulating in the blood. The character of the albu- 
minuria will largely depend upon the intensity of the intoxication; 
in other words, upon the amount of bacterial poison present at any 
one time in the blood. 

Notwithstanding statements to the contrary, albuminuria may be 
regarded as a constant symptom of typhoid fever, as has been defi- 
nitely demonstrated by Gubler and Robin. It is difficult to say why 
other observers have found albumin in only a comparatively small 
percentage of cases, but it is not unlikely that this is owing to a lack 
of uniformity in methods, it being presupposed also that questions 
of this kind can only be decided by daily examinations. According 
to Robin, the trace of albumin which is at times observed during 
the first week of the disease is an albumose, while later on serum 
albumin is constantly found; the amount increases with the inten- 
sity of the morbid process, and the highest figures are reached in fatal 
cases. The more severe the disease, the earlier does albumin appear 
in the urine, it being remembered, however, that reference is had 
only to those cases in which distinct renal changes are not demon- 
strable. Toward the termination of the fastigium the amount of 
albumin generally undergoes a certain diminution, and may even 
disappear entirely. This diminution, however, is only temporary, 
and in severe cases the albumin again increases in amount during 
the period of great variations in the temperature. In light cases 
an increased elimination also takes place at this stage, but is soon 
followed by a decrease, after which traces only can be demonstrated. 



448 THE URINE 

In some cases it disappears entirely, but it is rare, according to Robin, 
to meet with cases in which a trace at least does not reappear during 
convalescence. 

In light cases the albuminuria rarely persists longer than the fifth 
or eighth day of convalescence, and Robin even goes so far as to say 
that a relapse may be anticipated if the albuminuria does not disap- 
pear at that time. A limited number of personal observations have 
borne out the correctness of this view. In severe cases, on the other 
hand, the albumin persists for a variable length of time, and rarely 
disappears before the tenth day of convalescence. At times an 
increase is seen during convalescence when traces only have previously 
been observed. It is this form which the French generally speak of 
as colliquative albuminuria. While this is principally observed in 
typhoid fever, it is not unusual to meet with it during convalescence 
from various other acute diseases. Care must be taken not to con- 
found the albuminuria so frequently seen during convalescence from 
typhoid fever, referable to a pyelitis, with the form just described. 

From the following summary, Constructed from data given in 
Robin's 1 monograph on the urine of typhoid fever and other acute 
infectious diseases which may be associated with a typhoid condition, 
an idea may be formed of the occurrence of albuminuria, as well as 
of its degree of intensity in these diseases: 

Acute miliary tuberculosis: albumin is much less frequent than 
in typhoid fever; when present, it is rarely found in the abundance 
so characteristic of the fatal cases of the latter disease. 

Pneumonia: albumin is as uniformly present as in typhoid fever, 
and at times very abundant. 

Grippe: albumin is infrequent; present in about 20 per cent, of 
the cases, and only in traces. 

Herpetic fever: albumin never present in large amounts. 

Embarras gastrique: albumin rarely present. 

Adynamic enteritis of adults: albumin almost always present, but 
usually only in traces. 

Cerebrospinal meningitis: albumin in fairly large amounts. 

Malignant endocarditis: albumin very abundant in about 14 per 
cent., evident in 44 per cent., and traces in 42 per cent, of all cases. 

Acute articular rheumatism: albumin present in about 40 per 
cent. 

Rubeola: albumin usually absent in light cases, but present in the 
more severe and complicated forms. 

Intermittent fever: albumin variable. 

In a series of 799 cases of pneumonia reported from the Boston 
City Hospital, 2 albumin was found in 624 — i. e., in 78 per cent. It 

1 Urologie clinique de la fievre typhoide, Paris, 1877. 

2 Sears and Larrabee, Med. and Surg. Rep. of the Boston City Hospital 12th 
Series, Dec, 1901. 



CHEMISTRY OF THE URINE 449 

was noted that the death rate bore a direct ratio to the amount of 
albumin in the urine. 

In smallpox a trace of albumin is practically constant. Some- 
what larger amounts are found in about 30 per cent, of all cases. 
The albuminuria is most marked during the eruptive stage and 
then rapidly diminishes in intensity. More rarely it reaches its 
maximum during the suppurative fever stage, or during con- 
valescence. 1 

As the result of the examination of a large number of cases of 
plague Corthorn 2 arrived at the conclusion that no albumin f is found 
in only 14 per cent, of all cases. In cases ending in recovery the 
albuminuria never occurred later than the fourth day. 

In conclusion, it may be said that practically every acute febrile 
disease, even simple follicular tonsillitis, may be accompanied by 
albuminuria in the absence of definite changes affecting the renal 
parenchyma. Its occurrence in an individual case is probably 
dependent to a very large degree upon the intensity of the intoxica- 
tion. While it is generally an easy matter to distinguish between 
this form of albuminuria and that associated with distinct organic 
changes in the kidneys, considerable difficulty may at times be experi- 
enced; this question will be dealt with later on. 

4. Albuminuria referable to Circulatory Disturbances. 3 
— To this class belongs the albuminuria so frequently observed in 
cardiac insufficiency referable to valvular lesions, degeneration of the 
heart muscle from whatever cause, disease of the coronary arteries, 
etc., as well as in cases of impeded pulmonary circulation affecting 
the general circulation through the right heart, and, finally, in con- 
ditions associated with local circulatory disturbances, such as com- 
pression of the renal veins by a pregnant uterus, tumors, etc. It 
has been pointed out that febrile albuminuria also may, to a certain 
extent at least, be referable to such causes — i. e., an ischemia or 
hyperemia of the kidneys produced by an increased or diminished 
blood pressure. The albuminuria observed in cases of cholera 
infantum, the simpler forms of intestinal catarrh, and in cholera 
Asiatica particularly, are undoubtedly dependent upon such causes. 
The quantity of albumin found under these circumstances varies con- 
siderably, but rarely exceeds 0.1 to 0.2 per cent, unless the disease has 
advanced to a stage where distinct changes in the renal parenchyma 
have resulted. The occurrence of albuminuria after cold baths, as 
stated above, is regarded by many as a "physiological" phenomenon, 
but this view should be rejected, as there can be little doubt that this 
form is also referable to circulatory disturbances. 

1 Arnaud, " Albuminuric et lesions des reins dans la variole," Rev. d. med., 1898, 
vol. xviii, p. 392. 

2 "Albuminuria in Plague," Brit. Med. Jour., Sept. 14, 1901. 

3 Senator, loc. cit. 

29 



450 THE URINE 

5. Albuminuria referable to an Impeded Outflow of 
Urine. — Clinically, albuminuria referable primarily to an impeded 
outflow of urine from the kidneys is probably of more frequent 
occurrence than is generally supposed, and especially in women, in 
whom Kelly and others have demonstrated the frequent existence 
of ureteral stenoses. A complete blocking of the excretory duct, 
on the other hand, is rarely seen, but may be caused by the impac- 
tion of a renal calculus, the pressure of a tumor, or following cer- 
tain gynecological operations in which the ureter is accidentally caught 
in a suture, etc. It has also been suggested that the albuminuria 
of pregnancy may be due to a compression of a ureter, but it is more 
likely that other factors are here at play. 

6. Albuminuria of Hemic Origin. 1 — It was formerly supposed 
that Bright's disease was dependent upon certain abnormalities of 
the blood, and as a matter of fact this view has not only never been 
disproved, but is actually gaining ground from day to day. Accord- 
ing to Semmola, Bright's disease is primarily due to an abnormal 
power of diffusion on the part of the albumins of the blood, which 
are eliminated by the kidneys as waste material. As a result of the 
excessive amount of work thus done, definite renal changes are finally 
produced. According to his theory, then, the albuminuria is the 
primary factor in the causation of nephritis. Should this hypothesis 
hold good, Senator is correct in asserting that an albuminuria of 
functional origin, so to speak, must precede the occurrence of the 
nephritis proper. He, however, doubts the occurrence of a pre- 
nephritic albuminuria; but others have noted the occurrence of defi- 
nite renal changes which manifestly followed an apparently functional 
albuminuria (Da Costa). Further researches in this direction are 
urgently needed, and Semmola's view can at present only be regarded 
as an hypothesis. But even if such blood changes as those which 
Semmola suggests should not exist, there can be little doubt that 
true nephritis is dependent upon an acute or chronic dyscrasia of 
the blood, either in the sense of an abnormal mixture of the 
normal elements or of the presence of abnormal constituents. The 
same considerations undoubtedly also apply to various other forms of 
albuminuria, in so far as these are not the direct result of circulatory 
disturbances. 

Clinically, albuminuria of hemic origin is observed in various 
diseases of the blood, such as purpura, scurvy, leukemia, pernicious 
anemia, as also in cases of poisoning with lead and mercury, in 
syphilis, jaundice, diabetes, following the inhalation of ether and 
chloroform, etc. The albuminuria associated with an excessive 
elimination of uric acid and oxalic acid, and, according to 
personal observations, with an excessive elimination of organic 

1 v. Bamberger, loc. cit. 



CHEMISTRY OF THE URINE 451 

material in general, notably of urea, probably also belongs to this 
class. 

7. Toxic Albuminuria. — It has already been stated that the 
albuminuria of acute febrile diseases may, to a certain extent, be 
referable to a direct irritant action on the part of bacterial poisons 
upon the renal parenchyma. Poisoning with cantharides, mustard, 
oil of turpentine, potassium nitrate, carbolic acid, salicylic acid, tar, 
iodine, petroleum, phosphorus, arsenic, lead, antimony, alcohol, and 
mineral acids produces albuminuria. In all probability, however, 
the albuminuria here observed is referable not only to a direct irri- 
tant action upon the glandular epithelium of the kidneys, but also 
to circulatory disturbances. 

8. Neurotic Albuminuria. — It is claimed by some that albu- 
min, usually in small amounts, is eliminated in epilepsy after every 
attack, while others either deny its occurrence under such conditions 
or regard it as exceptional. In a number of cases in which I had 
occasion to examine urine voided after an attack, albumin was usually 
absent. It should be stated, however, that the seizures in these 
cases were comparatively slight, and that unfortunately an exam- 
ination for semen was not made in those urines in which traces of 
albumin were demonstrated. An examination of the urine voided by 
a patient, after having been in the epileptic state for more than forty- 
eight hours, showed the presence of a small amount of albumin 
associated with an enormous elimination of uric acid, as well as a 
large excess of urea. Semen was absent. 1 Nothnagel states that he 
could not demonstrate any regularity in the appearance of albumin. 
In some of his cases with major attacks there was no albumin; in 
others it appeared after every attack; in still others it was some- 
times present and at other times absent (in the same individual). 
At times it was found after a minor attack and was absent after a 
major attack (also in the same individual). 

Other observers have obtained similar results, so that we may 
conclude that albuminuria following epileptic seizures is rather the 
exception than the rule. When it does occur, its significance is 
essentially the expression of a certain grade of cyanosis during the 
attacks. 2 

A transient albuminuria has also been noted in cases of progressive 
paralysis, mania, tetanus, delirium tremens, apoplexy, migraine, 
Basedow's disease, brain tumor, etc. 

Although albuminuria may apparently be produced artificially by 
injuries affecting a certain area in the floor of the fourth ventricle 
analogous to the production of glucosuria (see Glucosuria), it would 
probably be going too far to assume the existence of a certain spe- 

1 M. Huppert, Virchow's Archiv, 1874, vol. lix, p. 305. 

2 Nothnagel, Ziemssen's Handbuch, 1877, vol. xii, p. 179. Binswanger, Noth- 
nagel's spec. Pathol, u. Therap., vol. xii, p, 235 (literature). 



452 THE URINE 

cific centre, stimulation of which causes the appearance of albumin 
in the urine. While the influence of the nervous system in prevent- 
ing the passage of albumin through the glomeruli under normal 
conditions is undoubted, it would appear more likely that the albu- 
minuria following injuries to the central nervous system is referable 
to circulatory disturbances in the kidneys secondary to lesions of 
the brain, and especially of the medulla. The albuminuria observed 
in certain neurotic individuals, on the other hand, is probably more 
frequently associated with metabolic abnormalities, and is of hemic 
origin. 

9. A Digestive Albuminuria has also been described. 1 It may 
follow the ingestion of excessive amounts of cheese, eggs — particu- 
larly when taken raw — beef, etc. Specially interesting is the form 
which follows the ingestion of excessive amounts of egg albumen. 
Ordinarily the consumption of a moderate amount of such albumen 
does not lead to albuminuria, while in cases of nephritis an already 
existing albuminuria is increased. But it has also been noted that 
even in individuals with apparently healthy kidneys, the ingestion 
of an excessive amount of egg albumen may call forth albuminuria, 
and it is possible in both cases to demonstrate the presence in the 
urine of both egg albumen and blood albumin. 

To examine into this question the individual is given from four to 
eight raw eggs on an empty stomach in the morning for two to four 
days. His diet otherwise is as usual. The urine is collected at 
intervals of from two to three hours. If the ingestion of such an 
amount of egg albumen leads to albuminuria, this usually occurs 
after about four hours, and reaches its maximum intensity two hours 
later. Casts are not found (Jnouye). 

The albuminuria in question, so far as the egg albuminuria goes, 
is undoubtedly owing to the fact that a certain amount of egg albu- 
men is absorbed as such from the gastro-intestinal canal and is subse- 
quently eliminated as foreign material. In what manner, however, 
the egg albuminuria may be responsible for the accompanying serum 
albuminuria is more difficult to explain. 

Of the albuminuria which follows excessive indulgence in cheese 
and beef but little is known. Bearing in mind that the albumin- 
uria very often follows the ingestion of such articles almost immedi- 
ately, and before they have become absorbed, it is hardly justifi- 
able to refer this form to the existence of a hyperalbuminosis. It 
would appear more rational, as Senator has suggested, to think 
of reflex vasomotor or trophic changes affecting the kidneys; while 
in other cases, in which the albuminuria does not follow the inges- 
tion of such articles of food immediately, it is quite probable that 

1 Ascoli, "Ueber d. Mechanismus d. Albuminuric durch Eiereiweiss," Munch. 
med. Woch., 1902, No. 10. Jnouye, " Ueber alimentare Albuminuric," Deutsch, 
Arch. f. klin. Med., 1902, vol. lxxv, p. 378. 



CHEMISTRY OF THE URINE 453 

it may be dependent upon certain metabolic abnormalities affecting 
the normal composition of the blood. 

In the account thus given of the occurrence of albuminuria and 
its possible causes, reference has been had to only a purely renal albu- 
minuria. It should be remembered, however, that the origin of the 
albumin may often be extremely difficult to determine, as albuminous 
material, such as blood and pus, may become mixed beyond the 
glandular portion of the kidneys with what would otherwise have 
been a perfectly normal urine, and that such an admixture may take 
place not only in the ureters, the bladder, and the urethra', but even 
in the pelvis of the kidney. 

The term accidental albuminuria is applied to a condition in which 
albuminous material becomes mixed with a urine beyond the kidneys, 
as in cases of cystitis and urethritis, or whenever semen has entered 
the urine while the renal urine proper is free from albumin. An admix- 
ture of pus, blood, lymph, or chyle may, however, also occur in the 
kidneys, when the albuminuria is termed accidental renal albuminuria, 
an example of which is frequently seen in the slight degree of albu- 
minuria referable to pyelitis during convalescence from typhoid 
fever. By a mixed albuminuria and a mixed renal albuminuria, on 
the other hand, we are to understand conditions in which the source 
of the albumin is twofold, renal and extrarenal in the first instance, 
parenchymal and extraparenchymal in the second, examples being the 
albuminuria of cystitis combined with nephritis and pyelonephritis, 
respectively. 

It is manifest, of course, that in every instance in which albumin 
is found in the urine its origin should be ascertained. While this 
question is usually readily decided by a microscopic examination 
of the urine, considerable difficulty may occasionally be experienced. 
It is a well-known fact that in the urine of women a trace of albu- 
min may frequently be detected, which is not due to any lesion of 
the urinary organs, but to an admixture of vaginal discharge, of 
blood during the process of menstruation, and, in married women, 
of semen. Whenever, therefore, doubt is felt as to the origin of the 
albumin, the specimen for examination should be obtained by the 
catheter, care being taken previously to cleanse the vulva. In men 
albumin may be referable to a gonorrheal urethritis. In such cases 
it is well to let the patient flush out his urethra first, and to make 
use for examination of the portion last voided. Very often, how- 
ever, the conditions are more complex, it being uncertain whether 
the albumin is referable to the presence of pus only, or whether its 
origin is in the renal parenchyma. In such cases, as in cystitis, 
pyelonephritis, etc., a careful microscopic examination and enumer- 
ation of the pus corpuscles with the Thoma-Zeiss instrument are 
called for, and will in the majority of instances decide the question. 
Generally speaking, the amount of albumin found in uncomplicated 



454 THE URINE 

cases of cystitis does not exceed 0.15 per cent., while in cases of 
pyelitis of the same intensity the amount of albumin is from two to 
three times as large. 

Of late, attention has repeatedly been drawn to the occasional 
presence in the urine of an albuminous body which is soluble in 
acetic acid, and which Patein regards as a modification of common 
serum albumin. It has thus far been observed in only 8 cases, 
viz., twice in chronic nephritis, three times in eclampsia, once in a 
cystic kidney, once in tonsillitis following an injection of diphtheria 
antitoxin, and once in a pregnant woman in whom typhoid fever 
developed. I should suggest that the substance be spoken of as 
Patein' s albumin 1 until its chemical identity has been established. 
The term acetosoluble albumin is, of course, likewise admissible. 

So far as the amount of albumin is concerned, which may be 
eliminated in the twenty-four hours, an excretion of less than 2 grams 
may be regarded as insignificant, 6 to 8 grams as a moderate amount, 
and 10 to 12 grams or more as excessive. An excretion of 20 to 30 
grams is exceptional. 

Serum Globulin. — It has been pointed out that in cases of amyloid 
degeneration of the kidneys serum globulin is found in the urine 
together with serum albumin in large amounts, and, according to 
Senator, a ratio between the two albumins of 1 to 0.8 to 1.4 may be 
regarded as a fairly constant symptom of the disease, and of diag- 
nostic importance. There seems to be no doubt, however, that 
serum globulin occurs in the urine, although in much smaller quan- 
tities than in the disease mentioned, whenever serum albumin is 
eliminated. 2 

A most remarkable instance of globulinuria has been recorded 
by Noel Paton, 3 in which the globulin separated out in crystalline 
form and was found in extraordinarily large quantity, amounting on 
one day to 70 grams. 

Albumoses. — Albumoses have frequently been encountered in the 
urine, but are probably more frequently overlooked, as the bodies 
in question are not precipitated on boiling. 

Albumosuria is observed under a great variety of conditions. 
It is thus noted in association with large accumulations of pus within 
the body, and there can be little doubt that the albumosuria is in 
such instances referable to a disintegration of the pus corpuscles 
and a resorption of the resulting albumoses. This form has hence 
been termed pyogenic albumosuria. It is principally observed during 
the stage of resolution in cases of croupous pneumonia; in associa- 

1 Patein, "Acetosoluble Albumin in the Urine," Compt.-rend. de PAcad. des 
sci., 1889. Coplin, Phila. Med. Jour., 1899, p. 957. 

2 Edlefsen, Deutsch. Arch. f. klin. Med., vol. vii, p. 67. Senator, Virchow's 
Archiv, vol. lx, p. 476. Petri, Diss., Berlin, 1876. 

3 B. Bramwell and N. Paton, Laboratory Reports of the Royal College of 
Physicians, Edinburgh, 1892, vol. iv, p. 47. 



CHEMISTRY OF THE URINE 455 

tion with pyothorax, and in cases of epidemic cerebrospinal menin- 
gitis, as contrasted with the tuberculous form. A hepatogenic form 
is noted in connection with diseases of the liver, notably acute yel- 
low atrophy. Of its origin, however, nothing is known. Formerly, 
when the condition was looked upon as a peptonuria, and when it 
was thought that peptones were retransformed into native albumins 
in the liver, the "peptonuria" was explained upon the assumption 
that the liver had lost this power, and that the " peptones" accumu- 
lated in the blood, and were consequently eliminated in the urine. 
At the present day this view is no longer tenable. 

An enterogenic form of albumosuria has been noted in various 
diseases of the intestinal tract, such as typhoid fever, tuberculous 
ulceration, carcinoma, etc.; and it is possible that in these cases the 
albumoses are either directly absorbed from disintegrating pus, or 
that the intestine perhaps has in part lost the power of preventing 
the resorption of albumoses as such into the blood. 

A histogenic or hematogenic origin has been ascribed to the albu- 
mosuria which is seen in cases of scurvy, in dermatitis, in various 
forms of poisoning, during the puerperal period and pregnancy, par- 
ticularly following the death of the fetus, in various psychoses, in 
cases of carcinomatosis, acute yellow atrophy, etc. 

A renal or vesical form of albumosuria is further noted in which 
the albumoses are derived from contained albumins, owing either to 
the presence of the common proteolytic ferments of the urine or to 
bacterial action, as in decomposing albuminous urines. 

Aside from the conditions already mentioned, albumosuria has 
been observed in various septic conditions, in diphtheria, measles, 
scarlatina, acute articular rheumatism, mumps, malaria, phthisis; 
further, in association with leukemia, nephritis, puerperal parame- 
tritis, endocarditis, caries, pleurisy, heart disease, apoplexy, myxedema, 
carcinomatous peritonitis, in pneumonia, at the height of the disease 
and before resolution has set in, in liver abscess, etc. 

In the differential diagnosis of suppurative meningitis a positive 
albumose reaction, according to Senator, speaks strongly in favor of 
the existence of this disease. In support of this view he cites the 
case of a young man, the subject of a median otitis of long standing, 
in which symptoms pointing to a meningitis — viz., fever, headache, and 
pains in the neck — were present, but in which no albumosuria was 
found to exist, and in which an operation revealed the presence of 
a cholesteatoma. A digestive form of albumosuria has recently been 
described, in which albumoses appear in the urine after their inges- 
tion in large quantities, and it is claimed that this is observed only 
in cases of ulcerative disease of the intestinal tract. Only a positive 
result, however, is of value. 

Very frequently albumosuria accompanies albuminuria, a condi- 
tion which Senator has termed mixed albuminuria, and it is interest- 



456 THE URINE 

ing to note that the albumosuria may alternate with the albuminuria, 
and may precede or follow the latter. In any case in which albu- 
moses can be demonstrated in the urine the appearance of albumin 
should accordingly be anticipated. 

In all cases of albumosuria the amount of albumose that appears 
in the urine is relatively small, and as a rule cannot be demonstrated 
by the biuret test when applied directly to the native urine. On the 
contrary, it is necessary to isolate the substance more or less definitely 
before deductions can be drawn as to its presence or absence. 

Literature. — Hofmeister, Prag. med. Woch., 1899, vol. v, pp. 321 and 325. 
v. Noorden, Lehrbuch d. Path. d. Stoffwechsels, Hirschwald, Berlin, 1893, p.2l5. 
Senator, Deutsch. med. Woch., 1895, vol. xxi, p. 217. Stadelmann, Untersuch- 
ungen iiber Peptonurie, Bergmann, Wiesbaden, 1894. v. Jaksch. Prag. med. 
Woch., vol. v, pp. 292 and 303, and vol. vi, pp. 61, 74, 86, 133, 143; Zeit. f. klin. 
Med., 1883, vol. vi, p. 413. Krehl u. Matthes, Arch. f. klin. Med., 1895, vol. xlv, 
p. 54. Maixner, Zeit. f. klin. Med., 1884, vol. viii, p. 234. Fischel, Arch. f. 
Gynak., 1884, vol. xxiv, p. 27. v. Jaksch, Prag. med. Woch., 1895, vol. xx, p. 
430. Katz, Wien. med. Blatter, 1890, vol. xiv. L. v. Aldor, Berlin, klin. Woch., 
1899, pp. 765 and 785. 

Bence Jones' Albumin. — In association with the occurrence of mul- 
tiple myeloma of the bones, notably when affecting the thoracic skele- 
ton, a peculiar albuminous body may be found in the urine, which 
is apparently pathognomonic of the disease in question. It is to be 
noted, however, that cases have also been reported in which the 
substance was absent, so that a positive result only is of value. It 
was first observed by Bence Jones, and has heretofore been regarded 
as an albumose. From the researches of Magnus Levy and my 
own investigations, however, it appears that the substance is in 
reality a true albumin, as it yields a proto-albumose on peptic diges- 
tion; but it differs from all known albumins in its relative solu- 
bility on boiling, and in the readiness with which it dissolves in 
dilute ammonia after precipitation with alcohol. Like casein, it 
contains no hetero-group, but is distinguished from it by the pres- 
ence of a carbohydrate radicle and the probable absence of phos- 
phorus. It is crystallizable, and may occur in the urinary sediment 
in the form of typical spheroliths. 

The amount of the substance which may be found in the urine is 
variable. Some observers have noted an elimination of from 0.25 
to 6.0 pro mille, while others report much larger quantities. In 
Bence Jones' case the elimination rose on one occasion to 6.7 per 
cent., corresponding to a total output of 70 grams in the twenty- 
four hours — i. e., to nearly as much as the entire amount of the 
albumins of the blood plasma. 

As regards the origin of the albumin nothing definite is known, 
but there is reason to suppose that it is not derived from the mye- 
lomatous tissue as such. We may imagine, however, that through 
the agency of the cells of the abnormal tissue, viz., their products 



CHEMISTRY OF THE URINE 457 

of metabolism, the normal transformation of the ingested albumins 
into tissue albumins is impeded, resulting in the production of the 
substance in question, which is then eliminated as foreign matter. 

As the diagnosis of myeloma, in its early stages at least, is alto- 
gether dependent upon the demonstration of the albumin in question, 
a special examination should be made in this direction in all cases 
of obscure bone pain, as also in obscure cases of anemia, since Ell- 
inger has shown that at times the disease may take its course with- 
out the occurrence of local symptoms, while a marked anemia may 
exist. 

Of special interest in this connection is the fact that Ziilzer claims 
to have succeeded in bringing about the appearance of Bence Jones' 
albumin in the urine of animals by feeding with pyrodin, which is 
known to be a distinct hemolytic poison. 

It has been recorded by several observers that the Bence Jones albu- 
minuria was accompanied by ordinary albuminuria. In no case, 
however, was the presence of common albumin established in a satis- 
factory manner, and it appears to me that its presence was merely 
assumed, whenever the urine did not clear entirely on boiling (see 
tests). This is unwarrantable, as it is now well known that the Bence 
Jones albumin itself, after being precipitated by heat, may not alto- 
gether dissolve on boiling. In two such cases, where one might have 
been led to assume the existence of ordinary albumin, I could demon- 
strate conclusively that this was not present. I should recommend 
that in all such cases the urine be carefully and slowly heated to 56° 
C, and maintained at that temperature until no more albumin sepa- 
rates out and that on cooling it be filtered. The filtrate can then be 
tested as usual for common albumin, either by heat or other tests, 
and I think that it will be found that common albumin is not present. 
That the two conditions may occur together is of course a priori pos- 
sible, but in the previously recorded cases no satisfactory evidence 
has been brought forward to show that this did occur. 

Literature. — Bence Jones, Med. and Chir. Trans., 1850, vol. xxxiii; and Phil. 
Trans. Royal Soc. of London, 1848. Kiihne, "Ueber Hemialbumose im Harn/' 
Zeit. f . Biol., vol. xxix, p. 209. Ellinger, " Ueber d. Vorkommen d. Bence Jones'- 
schen Korper im Harn," Arch. f. klin. Med., 1898, vol. lxii, p. 255. Magnus 
Levy, Zeit. f. phvsiol. Chem., 1900, vol. xxx, p. 200. Hamburger, Johns Hopkins 
Hosp. Bull., Feb., 1901. Ziilzer, Berlin, klin. Woch., 1900, p. 894. C. E. Simon, 
Amer. Jour. Med. Sci., 1902, vol. cxxiii, p. 954. 

Peptonuria. — To judge from recent investigations by Ito, 1 
true peptone in the sense of Kiihne, may occur in the urine 
under pathological conditions. He obtained positive results in 
pneumonia, in advanced cases of phthisis, in ulcer of the stomach, 
and in several women after childbirth. The reaction was most 

1 " Ueber d. Vorkommen v. echtem Pepton im Harn," Deutsch. Arch. f. klin. 
Med., 1901, vol. lxxi, p. 29. 



458 THE URINE 

intense in the pneumonia cases; it appeared already before resolu- 
tion occurred, and disappeared a few days after the crisis. In the 
parturient women no reaction was obtained if the examination was 
delayed until after the tenth day. It is noteworthy that in the 
cases examined by Ito the peptonuria was always associated with the 
presence of albumoses (deutero-albumoses), and that the peptone was 
present in still smaller amount than the albumoses. 

Hemoglobin (Methemoglobin) . — Under normal conditions the dis- 
integration of the red blood corpuscles which is constantly taking 
place in the body never results in such a degree of hemoglobinemia 
as to be followed by an elimination of hemoglobin in the urine. 
Whenever the destruction of red corpuscles is so extensive, how- 
ever, that the liver is unable to transform into bilirubin all the blood- 
coloring matter set free, hemoglobinuria occurs. While these factors, 
then — i. e., an excessive destruction of the red blood corpuscles and 
an insufficiency on the part of the liver — must be regarded as explain- 
ing every case of hemoglobinuria, our knowledge of the ultimate 
causes of such excessive disintegration, as well as the manner in 
which these operate, is limited. Formerly the term hematinuria was 
applied to this condition. It was shown, however, that the pigment 
eliminated is in reality not hematin, but usually methemoglobin, and 
only at times hemoglobin, so that the term hemoglobinuria is also 
ill chosen. 

Most common is the hemoglobinuria produced by certain poisons, 
such as potassium chlorate, arsenious hydride, hydrogen sulphide, 
pyrogallic acid, naphtol, hydrochloric acid, tincture of iodine, car- 
bolic acid, carbon monoxide, etc., and also by morels (Helvella 
esculenta). 

Quite familiar is the hemoglobinuria observed following trans- 
fusion of the blood of animals into man, such as that of the calf 
and lamb; also the form seen in extensive burns and in insolation. 

While hemoglobinuria may occur in the course of any one of the 
specific infectious diseases, such as scarlatina, icterus gravis, variola 
hemorrhagica, typhoid fever, yellow fever, etc., it is said to be espe- 
cially frequent in cases of malarial intoxication. This view is not 
accepted by many; Osier, among others, believes that it has fre- 
quently been confounded with malarial hematuria. I have never 
seen an instance of malarial hemoglobinuria, and believe that in 
our more temperate zones it scarcely ever occurs. Bastianello 
asserts that it is likewise rare in Italy, but more common in Sicily 
and Greece, and very common in the tropics. According to the 
same observer, hemoglobinuria occurs only in infections with the 
estivo-autumnal parasite. A hemoglobinuria due to quinine is like- 
wise said to exist, but is certainly rare, excepting in patients who 
are suffering or have recently suffered from malarial fever. I have 
seen but one instance of hemoglobinuria following the ingestion of 






CHEMISTRY OF THE URINE 459 

quinine. To judge from the literature upon the subject, there can be 
no doubt that syphilis may under certain conditions be a potent fac- 
tor in the production of hemoglobinuria. This appears to be par- 
ticularly true of those cases of so-called paroxysmal hemoglobinuria 
in which bloody urine is voided from time to time, the attacks being 
frequently preceded by chills and fever, so as closely to simulate 
malarial fever. Other factors, also, notably cold, appear to be con- 
cerned in the production of this form. 

The occasional occurrence of hemoglobinuria in cases, of Ray- 
naud's disease, coincident with attacks of an epileptiform character, 
has been referred to in the chapter on the Blood. 

Hemoglobinuria has been observed in a case of leukemia com- 
plicated by icterus. 

Finally, an epidemic hemoglobinuria has been described as occur- 
ring in the newborn associated with jaundice, cyanosis, and nervous 
symptoms; of its causation we are in ignorance. 

While hemoglobinuria is rather uncommon, hematuria is frequently 
observed, and will be considered later on, as its recognition is not 
dependent upon the demonstration of the albuminous body, "hemo- 
globin," alone in the urine, but upon the presence of red corpuscles, 
which in hemoglobinuria are either absent or present in only very 
small numbers. 

Literature. — Hemoglobinuria: Rosenbach, Berlin, klin. Woch., 1880, vol. 
xvii, pp. 132 and 151. Ehrlich, Zeit. f. klin. Med., 1881, vol. iii, p. 383. Boas, 
Arch. f. klin. Med., 1885, vol. xxxii, p. 355. Kobler u. Obermaver, Zeit. f. klin. 
Med., 1888, vol. xiii, p. 163. 

Fibrin. — The occurrence of fibrin in the urine presupposes the 
presence of fibrinogen and a fibrinogenic ferment. It is seldom 
seen. According to Neubauer and Vogel, the fibrin may occur 
either as coagulated fibrin or in solution. In the former condition 
it is at times observed in the form of blood coagula, when its signifi- 
cance is essentially the same as that of hematuria in general, although 
it must be remembered that the usual form of hematuria is not asso- 
ciated with the presence of coagula. Colorless coagula of fibrin are 
seen in cases of chyluria or diphtheritic inflammation of the urinary 
passages. On the other hand, urines containing fibrinogenic material 
in solution are likewise seen but rarely, and are characterized by the 
fact that fibrinous coagula separate out only on standing, when they 
usually cover the bottom of the vessel; but at times they may change 
the entire bulk of urine into a gelatinous mass. This condition like- 
wise is essentially observed in cases of chyluria, but may possibly also 
occur in association with nephritis. Lostorfer 1 has reported an 
instance of this kind, in which fibrinous coagulation took place in the 
clear urine, which contained much albumin, but no blood. Post- 

1 Wien. klin. Woch., 1903, No. 7. 



460 THE URINE 

mortem chronic inflammatory changes and amyloidosis of the kidney 
were found, while the urinary passages proper were intact. 

Nucleo-albumin. — The question whether or not nucleo-albumin is 
a normal constituent of the urine is still under dispute. Personal 
investigations have led me to the conclusin that with complicated 
methods and large amounts of urine — from 5 to 25 liters — it is 
always possible to demonstrate its presence both under physio- 
logical and pathological conditions. With the usual tests and 
smaller amounts of urine, however, negative results only are obtained 
in strictly normal individuals. According to my experience, tri- 
chloracetic acid, with which Stewart 1 claims to have obtained posi- 
tive results in every one of the 150 normal urines which he ex- 
amined, does not precipitate nucleo-albumin when this is present 
in normal amounts. A nucleo-albuminuria recognizable by the avail- 
able tests does not exist under normal conditions. Even under 
pathological conditions nucleo-albumin is by no means always found. 
Sarzin 2 thus was unable to demonstrate its presence in 200 cases which 
he examined in Senator's clinic. Citron 3 arrived at similar results, 
and of several thousand urines which I have examined in this direc- 
tion positive results were obtained in only a small percentage of cases. 
It is essentially met with in diseases which directly or indirectly 
involve the integrity of the epithelial lining of the uriniferous tubules 
or of the bladder. It has thus been frequently found in cases of acute 
nephritis and associated with febrile albuminuria, although its pres- 
ence even then is not constant. In chronic nephritis it is more fre- 
quently absent than present. In cases of renal hyperemia and cystitis 
the results are variable. In 32 icteric urines Obermayer 4 obtained 
positive results without exception, and it appears that in leu- 
•kemia nucleo-albumin is also quite constantly present. During 
the administration of p\rogallol, naphtol, corrosive sublimate, tar 
preparations, arsenic, etc., as well as in cases of poisoning with aniline 
and illuminating gas, large amounts of the substance may be found. 

According to my experience, nucleo-albumin is frequently ob- 
tained in cases of so-called functional albuminuria, and it is not 
uncommon to find that this is still present when serum albumin 
and serum globulin can no longer be demonstrated, even with the 
trichloracetic acid test. Nucleo-albuminuria may thus exist inde- 
pendently of the presence of the more common forms of albumin. 
This observation has also been made by Strauss, who found nucleo- 
albumiu only in several cases of cystitis, in one case of chronic inter- 
stitial nephritis, and in one case of emphysema pulmonum with renal 
hyperemia. 

1 Med. News, 1894. 

2 Ueber Nucleo-albuminausscheidimg, Diss., Berlin, 1894. 

3 Ueber Mucin im Harn, Diss., Berlin, 1886. 

4 Centralbl. f. klin. Med., 1892, vol. xiii, p. 1. 



CHEMISTRY OF THE URINE 46 i 

The existence of a hematogenic form of nucleo-albuminuria has 
thus far not been satisfactorily demonstrated. It has been assumed 
that its presence indicates increased epithelial desquamation in some 
portion of the urinary tract — in other words, that it is of cellular 
origin. Matsumoto, however, has shown that even though a urine 
containing numerous epithelial casts, renal epithelial cells, and leu- 
kocytes be allowed to stand for some time, a substance which can be 
precipitated with acetic acid either does not occur at all or only in very 
small quantity. He has rendered it very probable that the substance 
which can be precipitated from pathological urines by means of acetic 
acid is largely fibrinogen and euglobulin. He adds that nucleo-albumin 
may be present simultaneously, but in comparison to the other two 
substances it is of secondary importance and is rarely seen. 

Histon and Nucleohiston. — Kolisch and Burian 1 were able to dem- 
onstrate the presence of histon in a case of leukemia in which it was 
constantly present. More recently Krehl and Matthes 2 claim to 
have isolated the same substance in various febrile diseases, such as 
acute peritonitis, following appendicitis, in croupous pneumonia, 
erysipelas, and scarlatina. It is an albuminous body, and was first 
discovered by Kossel in the red blood corpuscles of the goose. It 
exists in the leukocytes of human blood in combination with the acid 
leukonuclein, constituting the so-called nucleohiston of Lilienfeld. 

It is not clear in what manner the histonuria is produced; so much, 
however, seems certain, that it is not solely dependent upon increased 
destruction of leukocytes. 

Nucleohiston itself has been found in the urine in a case of pseudo- 
leukemia, by Jolles. 3 

Tests for Albumin. — The recognition of the various albuminous 
bodies which may occur in the urine is based partly upon their direct 
precipitation and partly upon color reactions when treated with cer- 
tain reagents. 

The number of tests which ha\e from time to time been sug- 
gested is large; many of them after a brief period of use have been 
discarded as useless or uncertain, while others have been employed 
only occasionally, and have not received the recognition which they 
deserve, from the fact that simpler tests exist, that they do not possess 
sufficient delicacy, or that in some instances it is too great. In 
the following pages no attempt is made to describe all of these tests, 
and attention will be directed only to those which are generally used, 
and which clinical experience has proved to be of value, precedence 
being given to those which have been longest in use. While some of 

1 "Ueber d. Eiweisskorper d. leukamischen Hams," etc. ; Zeit. f. klin. Med., 
vol. xxix, p. 374. 

2 "Ueber febrile Albumosurie," Deutsch. Arch. f. klin. Med., vol. liv, p. 508. 

3 Ber d. deutsch. chem. Gesellsch., vol. xxx, p. 172; Zeit, f. klin. Med., vol. 
xxxiv, p. 53. 



462 THE URINE 

these are applicable for demonstrating the presence of more than one 
form of albumin, special tests will also be described whereby the 
various albumins may be individually recognized. 

In every case the urine should be carefully filtered, so as to free 
it from any morphological elements, etc., present. To this end, it 
is generally sufficient to pass the urine through one or two layers 
of Swedish filter paper. Frequently, however, a clear specimen 
cannot be obtained in this manner; it is then advisable to shake the 
urine with burnt magnesia or talcum, or to mix it with scraps of 
filter paper, when it is filtered as usual. 

Tests for Serum Albumin. The Nitric Acid Test 1 (Plate XVIII). 
— The value of this test, properly applied, cannot be overestimated, 
as it is not only simple, but yields an amount of information that can 
otherwise be gained only with difficulty. Usually the student is 
advised to make use of a test-tube partially filled with urine, along 
the sides of which concentrated, chemically pure nitric acid is allowed 
to flow, so as to form a layer at the bottom of the tube, when in the 
presence of serum albumin a distinct white ring appears at the zone 
of contact between the two liquids (Heller's test). The pictures thus 
obtained cannot be compared, however, with those seen when the 
apparently trivial change is made of using a conical glass of about 2 
ounces capacity instead of the test-tube. About 20 c.c. of urine are 
placed in the glass, when 6 to 10 c.c. of nitric acid are added by inclin- 
ing the glass and allowing the nitric acid to flow down the sides. 
When this is carefully done the nitric acid forms a distinct zone 
beneath the urine. In the presence of albumin the white ring then 
appears, and varies in extent and intensity with the amount of 
albumin present. If now the contents of the glass are allowed 
to stand undisturbed — and if small amounts are present, the albumin 
appears on standing for a few minutes — it will be observed that the 
cloudiness gradually extends upward ; and if much albumin is present, 
it may be seen to rise into the supernatant liquid in the form of small, 
irregular columns. This appearance is possibly referable to the 
decomposition of uric acid by means of nitric acid, nitrogen and 
carbon dioxide being set free, which, rising to the surface in the 
form of small bubbles, carry the nitric acid upward; coming into 
contact with albumin in solution, this is then precipitated. 

In practically every urine on standing for a few minutes, a fine 
ring appears in the clear urine above or separated from the albuminous 
ring by a distinct clear layer of urine (Plate XVIII). This ring 
has been generally ascribed to the presence of urates and in certain 
hospitals of Paris it was long customary to gauge the amount of uric 
acid by the rapidity with which it forms and its extent. For years 
I regarded this as an established fact, but I have convinced myself 

1 J. F. Heller, Arch. f. physiol. u. path. Chem. u. Micros., 1852, vol. v, p. 169. 
A. Robin, Urologie clinique de la fievre typhoide, Paris, 1877. 



PLATE XVIII 




Cold Nitric Acid Test. 

Albumin ring below; " urate" ring abov< 



CHEMISTRY OF THE URINE 463 

that no relation exists between this phenomenon and the amount of 
uric acid, as determined by one of the standard methods. Morner 
has expressed the opinion that the ring in question is not referable to 
urates at all, but is of a special albuminous character. Further 
researches in th's direction are needed. Usually the ring is fine and 
delicate, but at times the substance is present in large amounts and 
may simulate common albumin, by rapidly extending downward. 
Its clinical significance is not understood. 

Should more than 25 grams of urea be contained in a liter of the 
urine, an appearance like hoarfrost will be noted on the sides of the 
vessel, which is due to the formation of urea nitrate. Spangles of 
the same substance appear only in the presence of at least 45 grams; 
and if 50 grams or more of urea are contained in the liter, a dense 
mass of urea ni rate may be seen to separate out. 

Biliary urine, when treated with nitric acid containing a little 
nitrous acid, shows the color play referable to the action of nitric 
acid upon bilirubin. The production of the colors (red, yellow, 
green, blue, and violet) takes place from above downward, the green 
color being the most characteristic; in the absence of the latter the 
presence of biliary pigment may be positively excluded. The presence 
of albumin does not interfere, as the color play takes place beneath 
the albuminous disk. 

In normal urine a transparent ring is also obtained, presenting a 
peach-blossom red; the intensity of this may vary, however, from a 
faint rose to a pronounced brick color, and is referable to normal 
urinary pigment. In the presence of urobilin, on the other hand, 
this ring presents a distinct mahogany color. 

Indican is indicated by the appearance of a violet ring situated 
above that referable to the normal urinary pigment. Its intensity 
varies with the amount present, from a light blue to a deep indigo. 

The albumin ring at the zone of contact of the two fluids may be 
referable not only to the presence of serum albumin, but also of 
globulin and albumoses, while a negative reaction indicates the 
absence of these bodies. Should the precipitate caused by nitric 
acid consist of albumoses, it will clear up more or less, to reappear 
on cooling, the fluid at the same time assuming a markedly yellow 
color. The occurrence of a distinctly yellow color in the urine, 
moreover, which is only partially cleared upon the application of 
heat (and be it remembered that a much higher temperature is 
necessary for the solution of a precipitate referable to albumoses 
than of one due to urates), will indicate the existence of a mixed 
albuminuria — i. e., the presence of coagulable albumin and albumoses. 

Nitric acid may also cause a precipitation of certain resinous bodies, 
such as those contained in turpentine, balsam of copaiba and tolu, etc. 
If any doubt is felt, the mixture should be shaken with alcohol, 
when the precipitate caused by these substances is at once dissolved. 



464 THE URINE 

Nucleo-albumin, which is at times found in the urine, is also pre- 
cipitated by nitric acid, but need not occupy our at'ention at this 
place. From what has been said, it is manifest that the employment 
of the nitric acid test in the manner indicated furnishes much valuable 
information, and the adoption of the method as described not only by 
hospital students, but by general practitioners as well, cannot be too 
strongly urged. 

Boiling Test. — A few cubic centimeters of urine are boiled in a 
test-tube and then treated with a few drops of concentrated nitric acid, 
no matter whether a precipitate has occurred upon boiling or not. 
If albumin is present, this will separate out as a flaky precipitate, 
which consists of serum albumin frequently mixed with serum- 
globulin. It is true that albuminous urines will generally yield a 
precipitate on boiling alone; but it must be remembered that unless 
the reaction is decidedly acid a precipitation of normal calcium 
phosphate may occur, owing to the fact that the reaction of the urine 
upon boiling becomes less acid from the escape of carbonic acid held 
in solution. In urines presenting an alkaline or amphoteric reac- 
tion this is very frequently noted, and might give rise to confusion, 
as the precipitate due to calcium phosphate closely resembles that 
referable to albumin. In an alkaline medium, moreover, albumin 
may not be precipitated at all on boiling. Care must hence be taken 
to ensure a distinctly acid reaction, which is best accomplished by the 
addition of nitric acid, when a precipitate referable to phosphates is at 
once dissolved, while one due to albumin remains, and may even 
become more marked. The quantity to be added should usually be 
equivalent to about 0.05 to 0.1 per cent, of the volume of the urine. 
Under no condition should the acid be added before boiling, nor should 
the urine be boiled after its addition, as small amounts of albumin will 
otherwise be overlooked, owing to the fact that hot nitric acid dis- 
solves the precipitate to a certain degree. If, after the addition of 
the nitric acid the urine turns a distinct yellow, and if then upon 
cooling a white precipitate appears, the presence of albumoses may 
be inferred. Uric acid will cause no confusion, as this separates out 
only upon cooling, and then presents a dark-brown color. As in the 
case of the nitric acid test, so also here, a precipitation of certain 
resins is noted at times which may be recognized by their solubility 
in alcohol. Albumoses are also precipitated upon the application of 
heat, but such precipitates again dissolve when the temperature 
approaches the boiling point. 

Should acetic acid be used instead of nitric acid, great care must 
be taken to avoid an excess, as otherwise the albumin will be dis- 
solved. As this danger diminishes the greater the quantity of salts 
contained in the urine, it is advisable to treat the urine first with a 
few drops of acetic acid until a distinctly acid reaction is obtained, 
and then to add one-sixth its volume of a saturated solution of 



CHEMISTRY OF THE URINE 465 

sodium chloride, magnesium sulphate, or sodium sulphate, when 
upon boiling a precipitation of the albumin will occur. Carried 
out in this manner, the test is absolutely certain and will demon- 
strate even minimal amounts of albumin. If an equal volume of 
a saturated solution of common salt is added to the acidified urine, 
albumoses are also precipitated, but the precipitate dissolves on 
boiling. 

The Potassium Ferrocyanide Test. — A few cubic centimeters 
of urine are strongly acidified with acetic acid (sp. gr. 1.064) and 
treated with a few drops of a 10 per cent, solution of potassium 
ferrocyanide, when, in the presence of but little albumin, a faint 
turbidity, or, if much albumin is present, a flaky precipitate, is 
noted, which is best recognized by comparison with a tube contain- 
ing some of the pure filtered urine, both tubes being held against 
a black background, v. Jaksch advises the careful addition, by 
means of a pipette, of a few cubic centimeters of fairly concentrated 
acetic acid, to which a little potassium ferrocyanide has been added, 
when the albumin, as in Heller's test, is seen to form a ring at the zone 
of contact between the two fluids. Instead of potassium ferrocyanide, 
potassium platinocyanide may also be employed, and has the advan- 
tage that the test solution is colorless. Concentrated urines should 
be previously diluted with water. The presence of albumoses may 
be inferred if the precipitate disappears upon boiling, while a partial 
clearing up indicates the combined presence of albumoses and coag- 
ulable albumin. 

At times the addition of acetic acid by itself is followed by the 
appearance of a cloud in the urine, which may be due to urates or 
to urinary mucin (nucleo-albumin), as already mentioned. In such 
cases the urine should be refiltered, diluted with water, and the test 
again applied; nucleo-albumin will dissolve in an excess of the acid. 

The Trichloracetic Acid Test. 1 — This test is undoubtedly 
the most delicate of those so far described, but not so delicate that 
a trace of albumin or nucleo-albumin can be demonstrated in every 
urine. An experience based upon the examination of several thou- 
sand urines with this reagent warrants my speaking with a certain 
degree of confidence upon the subject. Very frequently it is pos- 
sible with this method to demonstrate albumin in urines in which 
the more common tests yield negative results, but in which tube 
casts may nevertheless be found upon microscopic examination. 
The test is applied as follows : By means of a pipette 1 or 2 c.c. of 
an aqueous solution of the reagent (sp. gr. 1.147) are carried to the 
bottom of a test-tube containing the carefully filtered urine, so as to 
form a layer beneath the urine. In the presence of albumin a white 
ring will be seen to form at the zone of contact between the two fluids, 

1 F. Obermayer, Wien. med. Jahrbiich, 1888, p. 375. D. M. Reese, Johns 
Hopkins Hosp. Bull., 1890. 
30 



466 THE URINE 

varying in intensity with the amount of albumin present. So far as 
the test for albumin is concerned, this reagent possesses an advantage 
over nitric acid in that the colored rings, which are so confusing 
to the inexperienced, are commonly not observed. Serum albumin, 
serum globulin, and albumoses are precipitated, the presence of the 
latter being recognized, as in the previous tests, by the fact that the 
precipitate disappears upon boiling and reappears on cooling. A 
cloud, referable to uric acid (?), also appears if this is present in exces- 
sive amounts, but disappears upon the application of gentle heat. 
A previous dilution of the urine, moreover, guards against its occur- 
rence. 

Other tests have also been suggested for the detection of albumin 
in the urine, such as the metaphosphoric acid test, the phenol, tannic 
acid, and picric acid tests, that with Tanret's reagent, phospho- 
tungstic and phosphomolybdic acids, Spiegler's reagent, etc. 

Of these, only the picric acid and Spiegler's test will be considered. 

Picric Acid Test. — The picric acid test is not applicable as a 
test for albumin as such, and is mentioned in this connection only 
because the same reagent is employed with Esbach's quantitative 
method. It is composed of 10 grams of picric acid and 20 grams 
of crystallized citric acid, dissolved in a liter of distilled water. If to 
this solution albuminous urine is added, the mixture is rendered 
turbid, and after some time a sediment which consists not only of 
albumins, but also of uric acid, kreatinin, and other extractives, will 
form at the bottom of the tube. (See Quantitative Estimation of 
Albumin.) 

Spiegler's Test 1 (Jolles' Modification). — The reagent consists 
of 10 grams of mercuric chloride, 20 grams of succinic acid, and 20 
grams of sodium chloride, dissolved in 500 c.c. of distilled water. 
The urine is acidified to the extent of 5 c.c. with 1 c.c. of dilute 
acetic acid (30 per cent.), then superimposed by means of a pipette 
upon 4 to 5 c.c. of the reagent when in the presence of albumin a 
distinct white ring appears at the zone of contact. On warming, the 
precipitate does not disappear. As mucin is precipitated by the 
acid, it is well in doubtful cases to use for comparison 5 c.c. of the 
acidified urine, diluted with 4 to 5 c.c. of water. Albumoses and 
nucleo-albumin are also thrown down, and in the presence of iodides 
mercuric iodide is precipitated; the latter is soluble in alcohol. 

Special Test for Serum Albumin. — Should it be desired, for 
any reason, to demonstrate serum albumin alone, the urine is ren- 
dered amphoteric or faintly alkaline with sodium hydrate, and is then 
saturated with magnesium sulphate in substance, in order to remove 
any globulin. The filtrate is rendered distinctly acid with acetic acid, 
when a flaky precipitate, appearing upon boiling, will indicate the 
presence of serum albumin. 

1 Wien. klin. Woch,, 1892, vol. v, p. 26. 



CHEMISTRY OF THE URINE 



467 



Patein's albumin differs from the common serum albumin in being 
soluble in acetic acid. 1 

Very often, as in the examination for sugar, it is necessary to 
remove any coagulable albumin that may be present, to which end 
the urine is rendered distinctly acid with acetic acid and boiled. An 
examination of the filtrate with potassium ferrocyanide, if the amount 
of acetic acid added was just sufficient, will then yield a negative 
result. 

Quantitative Estimation of Albumin. — For the quantitative esti- 
mation of albumin a large number of methods have been devised, 
which fact in itself is sufficient to indicate that the majority of them, 
at least, are unsatisfactory. 

Old Method by Boiling. — If comparative results only are desired, 
a definite amount of urine is boiled after acidifying with acetic 
acid; the albumin is allowed to settle for twenty-four hours. For 
this purpose Neubauer suggests the use of glass tubes measuring one- 
half to three-quarters of an inch in diameter, which are closed at the 
lower end with a cork. Ordinary test-tubes answer perfectly well, 
but care should be taken that the same quantity of 
urine is used in each case. The tubes are corked 
and kept for several days for comparison. The 
results, of course, express only the relative amount 
of albumin present, and it should be remembered 
that the error incurred may amount to as much as 
30 or even 50 per cent, of the quantity that is found 
by gravimetric analysis. This is owing to the fact 
that sometimes the albumin separates out in large 
flakes, and at other times in small flakes, and that 
the degree of precipitation is also influenced by the 
specific gravity of the supernatant urine. 

Esbach's Method. 2 — The reagent is composed of 
10 grams of picric acid and 20 grams of citric acid, 
dissolved in 1000 c.c. of distilled water. Special 
tubes, termed albuminimeters (Fig. 147), are em- 
ployed, which bear two marks, one, U, indicating 
the point to which urine must be added, and one, 
R, the point to which the reagent is added. The 
lower portion of the tube up to U bears a scale read- 
ing from 1 to 7, corresponding to the amount of 
albumin pro mille. The tube is filled to U with the 
filtered albuminous urine, and the reagent added until the point R is 
reached. The tube is closed with a stopper, inverted twelve times, 
and set aside for twenty-four hours. 

1 Patein, " Acetosoluble Albumin in the Urine/' Compt.-rend. de PAcad. des 
sci., 1889. Coplin, Phila. Med. Jour., 1899, p. 957. 

2 Guttmann, Berlin, klin. Woch., 1886, vol. xxiii, p. 117. 



Fig. 147— Esbach's 
albumini meter. 



468 THE URINE 

At the expiration of this time serum albumin, serum globulin, and 
albumoses, as well as uric acid and kreatinin, will have settled, when 
the amount pro mille in grams may be read off from the scale. A 
few precautions must be observed in order to obtain as accurate 
results as possible. The reaction of the urine should be acid, and 
if this is not the case acetic acid is added. Its specific gravity should 
not exceed 1.006 or 1.008, the proper density being obtained by dilut- 
ing with water. The amount of albumin in the specimen should 
not exceed 0.4 per cent.; if more be present, as determined by a pre- 
liminary test, the urine should be diluted. Most important, further- 
more, is the temperature of the room. This should be 15° C; varia- 
tions from this point are apt to give rise to inaccurate results, which, 
according to Christensen, may amount to 100 per cent, in the case 
of a deviation of only 5° C. It is thus clear that as generally em- 
ployed in the clinical laboratory the method will only give approxi- 
mate results. 

Gravimetric Method. — If accuracy is required the amount of albu- 
min must be determined gravimetrically as follows : A certain quan- 
tity of urine, after having been acidified with an amount of acetic 
acid sufficient to ensure complete precipitation of all albumin, is 
boiled; the albumin is then filtered off, dried, and weighed. For this 
purpose, 500 to 1000 c.c. of filtered urine should be available. A 
specimen of this, if already acid, is placed in a test-tube, in boiling 
water, until coagulation takes place, when it is further heated over 
the free flame and filtered. The filtrate is tested with acetic acid 
and potassium ferrocyanide. Should no albumin be thus demon- 
strable, the entire amount of urine is treated in the same manner, 
and requires no further addition of acetic acid. If, however, the 
test yields a positive result, it is apparent that the urine was not 
sufficiently acid. The entire volume is then treated with a 30 to 
50 per cent, solution of acetic acid, drop by drop, the mixture 
being thoroughly stirred and specimens tested from time to time, as 
described. When, finally, the urine remains clear or shows only a 
faint turbidity, 100 c.c. or less, according to the amount of albumin 
present, are first heated in boiling water until the albumin begins to 
separate out in flakes, and then brought to the boiling point over 
the free flame. The supernatant urine is decanted through a filter, 
which has been previously dried at 120° to 130° C. and accurately 
weighed, when the whole amount of the precipitate is brought upon 
the filter. Any albumin remaining in the beaker is detached from 
its sides by means of a glass rod tipped with a piece of rubber tubing, 
and collected by the aid of hot water. The entire precipitate is 
thoroughly washed with hot water until the washings no longer 
become turbid when treated with a drop of nitric acid and silver 
nitrate; in other words, until the chlorides have been removed. The 
precipitate is further washed with alcohol and finally with ether to 



CHEMISTRY OF THE URINE 469 

remove any fats that may be present, when it is dried at 120° to 130° 
C. to a constant weight. If still greater accuracy is required, the 
dried and weighed precipitate is incinerated to determine the amount 
of mineral ash in combination with the albumin, which is then de- 
ducted from the total weight. The most accurate results are obtained 
if not more than 0.2 to 0.3 gram of albumin is contained in the amount 
of urine employed. A smaller quantity than 100 c.c. should hence 
be used if a previous test with Esbach's albuminimeter shows a higher 
percentage. 

A glass-wool filter ensures a more rapid process of drying — twenty- 
four to thirty hours; but care must be had that this is properly pre- 
pared, so as to guard against a loss of the wool while washing. 

Method of Centrifugation. — This presupposes a constant speed, 
and hence an electrical centrifuge is a prerequisite, which is an 
objection to the general adoption of the method. Approximative 
results only are obtained. 

Test for Serum Globulin and its Quantitative Estimation. — To 
test for serum-globulin the urine is rendered alkaline by the addition of 
ammonium hydrate, any phosphates that may thus be thrown down 
being filtered off on standing. The urine is then treated with an 
equal volume of a saturated solution of ammonium sulphate, when 
the occurrence of a precipitate will indicate the presence of globulins. 
Ammonium urate may likewise separate out, but this occurs later. 

According to Paton, the following test may also be employed : The 
urine after having been rendered alkaline with sodium hydrate — 
any phosphates which may separate out are filtered off — is carefully 
poured down the side of a test-tube containing a saturated solu- 
tion of sodium sulphate, so as to form a layer above this, when in 
the presence of serum globulin a white ring will appear at the zone 
of contact. 

If a quantitative estimation of the globulin is to be made, the pre- 
cipitate thus obtained, after about one hour's standing, is collected 
on a dried and weighed filter, and washed thoroughly with a one- 
half saturated solution of ammonium sulphate until a specimen 
of the washings treated with acetic acid and potassium ferrocy- 
anide no longer gives a precipitate. It is then treated as directed 
in the method employed for the quantitative estimation of serum 
albumin. 

Tests for Albumoses. — A small amount of urine is strongly acidi- 
fied with acetic acid and treated with an equal volume of a saturated 
solution of common salt. In the presence of albumoses a precipitate 
occurs, which dissolves on boiling and reappears on cooling. If 
serum albumin also be present, which is usually the case, the hot 
liquid must be filtered. The albumoses are found in the filtrate and 
appear on cooling. If the hot filtrate, moreover, is rendered strongly 
alkaline with a solution of sodium hydrate, a red color develops upon 



470 THE URINE 

the addition of a very dilute solution of cupric sulphate (1 to 2 per 
cent.), added drop by drop (biuret reaction). On boiling with 
Millon's reagent a red color is also obtained. This reagent is prepared 
by dissolving 1 part of mercury in 2 parts of nitric acid of a specific 
gravity of 1.42, and diluting with 2 volumes of distilled water. 

Bang's Method. — 10 c.c. of urine are heated in a test-tube 
with 8 grams of finely powdered ammonium sulphate until the salt 
has been dissolved; the fluid is then boiled for a moment. The 
hot fluid is centrifugated for one-half to one minute, the supernatant 
fluid poured off, and the sediment stirred with alcohol in an agate 
mortar. The alcohol is poured off, and the residue dissolved in a 
little water; the solution is boiled and filtered, and the filtrate tested 
with sodium hydrate solution and cupric sulphate as described above. 
Should the urine be rich in urobilin — i. e., manifesting a well-marked 
fluorescence with zinc chloride and ammonia — it is best to extract 
the final aqueous solution with chloroform by shaking, and to pour 
off the supernatant fluid, when this is tested with cupric sulphate. 
In this manner it is possible to demonstrate the presence of albu- 
moses in a dilution of 1 in 4000 to 5000. Other constituents of the 
urine, with the exception of hematoporphyrin, do not interfere with 
the test. Should hematoporphyrin be present, however, which may 
be suspected if a red alcoholic extract is obtained, the urine must 
first be precipitated with barium chloride. The filtrate, which con- 
tains the albumoses, is then examined as described. 

If a centrifuge is not available, the urine is boiled with the ammo- 
nium sulphate, when a portion of the albumoses will remain on the 
sides of the tube as a sticky mass. This is washed with alcohol, 
and if necessary with chloroform, dissolved in water, and tested for 
biuret. 

The alcoholic extract may also be used for testing for urobilin. 
To this end it is only necessary to add a few drops of a solution 
of zinc chloride, when in the presence of urobilin a beautiful fluores- 
cence will be observed. The test is extremely delicate. 1 

Examination for True Peptone 2 (Polypeptids). — To demonstrate 
the presence of true peptone (in the sense of Kuhne) in the urine, 
about 300 c.c. of filtered acid urine are saturated on a water bath 
with ammonium sulphate at a temperature between 60° and 70° C. 
On cooling, the mixture is filtered, the filtrate is alkalinized with 
a dilute solution of sodium carbonate, again saturated between 60° 
and 70° C. with ammonium sulphate, filtered on cooling, the filtrate 
neutralized with very dilute acetic acid, again saturated with the salt 
between 40° and 50° C, and finally again filtered on cooling. The 
final filtrate is diluted with an equal volume of distilled water and 

1 E. Bang, "Eine neue Methode zum Nachweis d. Albumosen im Harn," 
Deutsch. med. Woch., 1898, p. 17. 

2 Ito, loc. cit 



CHEMISTRY OF THE URINE 471 

treated with a freshly prepared solution of tannic acid, which is 
added drop by drop, care being taken to avoid an excess. The 
precipitate is filtered off the next day, dried in the desiccator upon 
the filter, powdered, and covered in a porcelain crucible with a 
small amount of baryta-water to which a little finely powdered baryta 
is added. The mixture is placed on a boiling water bath for three 
minutes, and after one or two hours it is filtered. If necessary, the 
solution is decolorized with neutral lead acetate. The biuret test 
is finally applied, and if positive indicates the presence of peptone 
in the sense of Kuhne. 

Tests for Bence Jones' Albumin. — The presence of Bence Jones' 
albumin is usually discovered on slowly heating the urine* to the 
boiling point. It will then be noted that at a temperature of from 
50° to 60° C. a more or less intense, milky turbidity develops, which 
on subsequent boiling either disappears entirely or partially, and 
reappears on cooling. The degree to which the urine clears on. 
boiling differs in different cases. As I have just stated, the turbid- 
ity may disappear entirely; but, on the other hand, urines are met 
with in which even a partial clearing can scarcely be made out. 
This is apparently dependent upon the degree of acidity of the urine, 
the amount of mineral salts and of urea present, and probably also 
upon other and still unknown factors; it does not necessarily indicate 
that common albumin is simultaneously present. 

Upon the addition of a drop of nitric acid to a few cubic centi- 
meters of such urine a temporary turbidity develops, which disap- 
pears on shaking, but persists if a little more of the acid is added. 
If now the mixture is heated, the albumin first coagulates to a dense 
mass; on boiling, this dissolves, and after a while the liquid becomes 
almost entirely clear, while the turbidity returns, as before, on sub- 
sequent cooling. Similar reactions are obtained with all the common 
reagents for albumin. 

For its complete identification, the albumin should be isolated 
and further examined as follows : Large amounts of urine are pre- 
cipitated by the addition of one and one-half to two volumes of 
96 per cent, alcohol, or by treating with two volumes of a saturated 
solution of ammonium sulphate. In either event the total amount 
of albumin is thrown down. This is then washed with alcohol and 
ether, and dried over sulphuric acid. To purify the substance it is 
dissolved in boiling water, by the aid of a few drops of a dilute 
solution of sodium carbonate, and dialyzed to running and then to 
distilled water until free from mineral salts. It is then reprecipi- 
tated with alcohol (if necessary, after the addition of a drop or two 
of a dilute solution of hydrochloric acid), washed with absolute 
alcohol and ether, and dried. Thus purified, the albumin is prac- 
tically insoluble in distilled water or saline solution at ordinary tem- 
perature, and only sparingly so at the boiling point. In boiling 



472 THE URINE 

water, however, it dissolves with comparative ease after the addi- 
tion of a few drops of sodium carbonate solution. On neutraliza- 
tion no precipitate occurs if a sufficient amount of water is present. 
If such a neutral solution is heated, no change occurs; but if it is 
now acidified and a certain amount of salt added, the typical reaction 
appears on heating, viz., precipitation between 50° and 60° C. (even 
between 40° and 50° C. if a sufficient amount of salt is present), 
clearing on boiling, and reprecipitation on cooling. 

On digestion with pepsin-hydrochloric acid a proto-albumose is 
obtained among the early products of digestion, while a hetero- 
albumose is not formed. (See Bence Jones' Albumin, p. 456.) 

Boston's suggestion that the albumin in question can be recognized 
from its higher content of loosely combined sulphur, by qualitative 
examination, does not seem to the writer to be of value. 

Test for Nucleo-albumin. — It has been generally supposed that 
the substance which is precipitated on adding strong acetic acid 
to certain pathological urines, when diluted two or three times with 
water, is nucleo-albumin, the precipitate being soluble or largely so 
in an excess of the reagent. Matsumoto, 1 however, has recently 
pointed out that the substance which is precipitated in this manner 
is largely a mixture of fibrinogen (fibrinoglobulin) and euglobulin. 
Nucleo-albumin may be present at the same time, but it is rare, and 
its quantity in comparison to the two albumins mentioned insignifi- 
cant. 

To demonstrate the presence of nucleo-albumin, it is necessary to 
salt out the albumins with ammonium sulphate (half saturation is 
sufficient), and then to ascertain whether any precipitation occurs 
within the limits of precipitation of nucleo-albumin. Matsumoto 
gives these as 0.1 to 0.8 (lower limit) and 1.6 and 2.2 (upper limit). 
Its limits of precipitation are the lowest of the known albumins. 2 

Whether or not Ott's test 3 in the light of this work can still be 
relied upon as a test for the demonstration of nucleo-albumin may 
be questioned. It is conducted as follows: A few cubic centimeters 
of urine are treated with an equal volume of a saturated solution of 
common salt, when Almen's solution, which consists of 5 grams 
of tannic acid, 10 c.c. of a 25 per cent, solution of acetic acid, and 
240 c.c. of 40 to 50 per cent, alcohol, is slowly added. The develop- 
ment of a precipitate was regarded as evidence of the presence of 
nucleo-albumin. 

In order to remove nucleo-albumin from the urine, this is treated 
with neutral lead acetate, an excess of the reagent being avoided. 

1 Ueber d. durch Essigsaure ausfallbare Eiweissubstanz in pathologischen 
Harnen, Deutsch. Arch., 1902, vol. xxv, p. 398. 

2 Limit of precipitation of fibrinogen, 1.5 to 1.7 to 2.5 to 2.7; of fibrino- 
globulin, 2.2 to 2.9; of euglobulin, 2.8 to 3.3; of pseudoglobulin, 3.4 to 4.6. 

3 Centralbl. f. inn. Med., 1895, vol. xvi, p. 38. 



CHEMISTRY OF THE URINE 473 

Test for Hemoglobin. — The diagnosis of hemoglobinuria is based 
upon the demonstration of hemoglobin, viz., methemoglobin, in the 
urine in solution, in the absence of red corpuscles, or at least in the 
presence of only a very small number. 

Bloody urine is generally turbid, and may vary in color from bright 
red to almost black. 

Oxyhemoglobin, as such, can only be recognized by the spectro- 
scope; it gives rise to the appearance of two bands of absorption, 
situated between D and E, as described in the chapter on the 
Blood. 

The urine to be examined spectroscopically should be rendered 
feebly acid by means of acetic acid, and placed before the open slit 
of the spectroscope in a test-tube, beaker, or similar vessel, when the 
two bands of oxyhemoglobin will be seen, either at once or upon 
diluting with distilled water. If ammonium sulphide is added, the 
spectrum of reduced hemoglobin will be obtained. It must be 
remembered that more commonly the spectrum of methemoglobin 
is seen in cases of hemoglobinuria. 

The following tests, which will also indicate the presence of blood- 
coloring matter, cannot be employed to decide the nature of the 
pigment present, as methemoglobin and oxyhemoglobin will both 
react in the same manner. 

Heller's Test. 1 — A small amount of the urine, or still better a 
portion of the sediment, is made strongly alkaline with sodium hydrate 
and boiled. On standing, a deposit of basic phosphates forms, which 
in the presence of blood-coloring matter presents a bright-red color. 
This is referable to the formation of hemochromogen, as may be 
shown by spectroscopic examination. Thus controlled, the test is 
extremely sensitive, and still yields a positive result when the chem- 
ical test alone leaves one in doubt. 2 The deciding band is the first 
between D and E. Care should be had, however, that the solution 
is cold, as otherwise the hemochromogen is transformed into hematin 
in alkaline solution. At times, when the urine contains a large 
amount of coloring matter (bile pigment, etc.), it may be difficult to 
determine the exact color of the sediment. In such cases the sub- 
sequent examination with the spectroscope — the lensless instrument 
of Hering or that of Browning suffices — is invaluable. In the 
absence of such apparatus the procedure of v. Jaksch may be em- 
ployed. To this end the phosphatic deposit is filtered off and dis- 
solved in acetic acid, when if blood pigment is present the solution 
becomes red, the color gradually vanishing upon exposure to the air. 
The delicacy of the test is such that oxyhemoglobin can still be 
demonstrated in a dilution of 1 to 4000. 

1 Zeit. d. K. K. Gesellsch. d. Aerzte zu Wien, 1858, No. 48. 
V. Arnold, Berlin, klin. Woch., 1898, p. 283. 



474 THE URINE 

Donogany's Test. 1 — About 10 c.c. of urine are treated with 1 
c.c. of a solution of ammonium sulphide and the same amount of 
pyridin, when in the presence of blood a more or less intense orange 
color develops, especially if looked at from above, against a white 
background. In doubtful cases the examination is to be controlled 
by a spectroscopic examination of the resulting mixture. If blood 
pigment is present, the spectrum of hemochromogen is obtained. 
Should the ammonium sulphide and pyridin be old, a green or brown 
color is imparted to the urine, which changes to yellow upon the 
addition of ammonium hydrate. 

Test for Fibrin. — Fibrin usually occurs in the urine in the form 
of distinct clots, the nature of which may be determined by thor- 
oughly washing with water, when they are dissolved by boiling in a 
1 per cent, solution of soda or a 5 per cent, solution of hydrochloric 
acid. On cooling, this solution is tested as for serum albumin. 

Test for Histon. — The urine of twenty-four hours is first examined 
for albumin, and this removed if present. It is then precipitated 
with 94 per cent, alcohol, the precipitate washed with hot alcohol 
and dissolved in boiling water. Upon cooling, the solution thus 
obtained is acidified with hydrochloric acid and allowed to stand for 
several hours. During this time a cloudiness, referable to a large 
extent to uric acid, develops, which is filtered off, and the filtrate is 
precipitated with ammonia. The precipitate is collected on a small 
filter and washed with ammoniacal water until the washings no 
longer give the biuret reaction. It is then dissolved in dilute acetic 
acid and the solution tested with the biuret test; if this yields a 
positive result, and if coagulation occurs upon the application of 
heat, the coagulum being soluble in mineral acids, the presence of 
histon may be inferred. 

Carbohydrates. 

Glucose. — Through the researches of Wedenski, v. Udranszky, 
and others, 2 we know that traces of glucose may be encountered in 
the urine under strictly normal conditions. The amount, however, 
is extremely small, and special methods are necessary in order to 
demonstrate its presence. With the usual clinical tests normal urine 
is apparently free from sugar unless unduly large amounts have 
recently been ingested. In that event a certain amount of glucose 
is eliminated in the urine, constituting the so-called digestive gluco- 
suria of Claude Bernard. 3 

1 "Darstellung d. Haemochromogen als Reaction auf Blut," etc., Virchow's 
Arc hi v, vol. cxlviii, p. 234. 

2 A. Baumann, Br. d. Deutsch. chem. Ges., 1886, vol. xix, p. 3218. N. 
Wedenski, Zeit. f. physiol. Chem., 1889, vol. xiii, p. 122. K. Baisch, ibid., 1894, 
vol. xviii, p. 193, and 1895, vol. xix, p. 348. 

3 Compt.-rend. de PAcad. des sci., 1859, vol. xlviii, p. 673 



CHEMISTRY OF THE URINE 475 

The normal limit to the assimilation of glucose on the part of the 
body economy is subject to considerable variation. Some observers 
thus report that the ingestion of such large amounts as 200 and 250 
grams does not lead to glucosuria, while others have found sugar 
in the urine after the administration of 100 grams. In view of 
the possible relation existing between diabetes and a lowered limit 
to the assimilation of glucose in apparently normal individuals, or at 
least in persons in whose urine glucose cannot be constantly tlemon- 
strated, this question has created much interest within the last few 
years and has called forth a large amount of work. The major- 
ity of investigators are now in accord in regarding as abnormal a 
glucosuria that follows the ingestion of 100 grams of chemically pure 
glucose. 

The method usually employed in order to ascertain the power of 
assimilation for glucose on the part of an individual is the following : 

The patient receives 100 grams of glucose, in substance, dis- 
solved in 500 c.c. of water, on an empty stomach, and is instructed 
to pass his water hourly during the following four to five hours. 
During this time, moreover, no food is to be taken. The individual 
specimens, as well as the urine which has been passed during the 
night, are then tested with Trommer's and Nylander's tests, with the 
fermentation test, and with phenylhydrazin. A positive result, how- 
ever, is recorded only when sugar can be demonstrated with the fer- 
mentation test. 

Cane sugar and larger amounts of glucose have also been used; 
but it is better, on the whole, as Strauss has pointed out, to give 
glucose, and not to exceed the dose of 100 grams. 

Especially interesting are the results which have been obtained in 
various diseases of the liver, to which organ the important function 
of preventing an undue accumulation of sugar in the blood has been 
repeatedly ascribed. Bierens de Haen 1 thus reports that of 29 cases 
of various hepatic diseases he found sugar in 18 after the administra- 
tion of 150 grams of cane sugar; and v. Jaksch 2 claims to have ob- 
tained positive results in 15 cases of phosphorus poisoning out of 43. 
Strauss, 3 on the other hand, states that he found sugar in only 2 of his 
38 cases, and has collected 107 additional cases from the literature, 
in only 14 of which could sugar be demonstrated. If we add these 
together, we have 145 cases of various hepatic diseases, with negative 
results in 88.9 per cent. Referring to the contradictory results 
obtained, Strauss points out that these may have been accidental in 
part, but that the interpretation which has been offered by v. Jaksch 
and de Haen may not have been correct. It is thus possible that in 

1 "Ueber alimentare Glycosurie bei Leberkranken," Arch. f. Verdauungskrank., 
vol. iv, p. 4. 

2 "Alimentare Glycosurie," Prag. med. Woch., 1895, Nos. 27, 31, and 32. 

3 "Leber und Glycosurie," Berlin, klin. Woch., 1898, p. 1122. 



476 THE URINE 

his cases of phosphorus poisoning other factors besides the changes 
in the liver, such as the action of the poison upon the nervous system, 
etc., played a role, as a digestive glucosuria may also occur in con- 
nection with other forms of intoxication, as in fevers, following the 
administration of large doses of diuretin, in acute alcoholism, etc., 
in which the liver is not the only organ that is involved. Strauss 
further shows that great care must be exercised in the selection of 
the material for such investigations, and believes that errors referable 
to this source may have been incurred by Bierens de Haen. He thus 
cites 2 cases of hypertrophic cirrhosis, associated with delirium 
tremens, in which small amounts of sugar could be demonstrated in 
the urine a few days after recovery from the delirium, while shortly 
after negative results only could be obtained. The lowering effect 
of alcoholism upon the limit to the assimilation of glucose is a well- 
known phenomenon, and it would be erroneous to conclude that 
because alcoholism may call forth organic changes in the liver the 
digestive glucosuria in such cases must be referable to such alterations. 
Without entering further into the question at this place, it appears 
that diseases of the liver per se do not materially lessen the power of 
assimilation for glucose, and that other forces are at the disposal of 
the body to supply the glycogen-forming or retaining power of the liver 
when this becomes insufficient, and that these also may be at fault 
when a digestive glucosuria is observed in association with hepatic 
disorders. 

The association of digestive glucosuria with various diseases of 
the nervous system has been carefully studied by v. Jaksch, 1 Strum- 
pell, H. Strauss, 2 von Oordt, Geelvink, and Arndt. 3 From the work 
of these investigators it appears that digestive glucosuria is rarely 
seen in spinal diseases, and is decidedly more common in functional 
diseases of the central nervous system than in organic affections. 
Of 30 cases of tabes examined by Strauss, digestive glucosuria 
resulted in only 1 after the administration of 100 grams of glucose, 
and in that case a family history of diabetes existed. In 16 further 
cases examined by J. Strauss negative results were obtained. In the 
neuroses a positive result was noted in 42 out of 210 cases which I 
have been able to collect from the literature. Most frequently it is 
met with in the traumatic neuroses, in whch Strauss observed the 
phenomenon in 37.5 per cent, of his 40 cases; while in the non-trau- 
matic forms only 14.4 per cent, were insufficient in this respect. Of 
the organic diseases of the central nervous system, it appears that 
diffuse cerebral lesions referable to alcohol and syphilis are more likely 

1 Loc. cit. 

2 "Zur Lehre v. d. neurogenen u. d. thyreogenen Glycosurie," Deutsch. med. 
Woch., 1897, pp. 275 and 309. 

3 "Ueber alimentare Glycosurie bei Neuropsy chosen," Berlin, klin. Woch., 1898, 
p. 1085. 



CHEMISTRY OF THE URINE 477 

to give rise to this form of glucosuria than the more localized lesions. 
In general paresis digestive glucosuria is thus not uncommon (H. 
Strauss, Arndt), but it is only possible to draw definite deductions 
from the study of a large amount of clinical material. Small series 
like that of J. Strauss do not give a proper idea of actual conditions, 
as he, for example, obtained negative results in all of 10 cases. 

In his examination of 5 cases of idiocy and 23 cases of imbecility, 
J. Strauss obtained positive results in only 2 of the imbeciles after 
the administration of 100 grams of glucose; in both of the posi- 
tive cases the glucosuria was transitory and associated with the 
existence of nervous excitability. Bergenthal observed alimentary 
glucosuria in 6 cases out of 20. 

In Basedow's disease digestive glucosuria has also been noted in 
a large number of cases by Chvostek, Kraus and Ludwig, Strauss, 
Goldschmidt and Stern. Especially interesting in this connection is 
the fact that digestive glucosuria may be induced by the administra- 
tion of thyroid extract, viz., thyroidin or iodothyrin in apparently 
normal persons. Bettmann 1 thus noted glucosuria after the inges- 
tion of 100 grams of glucose in 12 of 25 healthy individuals who 
had been treated for a' week with the products in question. 

A digestive glucosuria is further observed in numerous febrile dis- 
eases, such as pneumonia, typhoid fever, acute articular rheumatism, 
scarlatina, tonsillitis, etc. The amount of sugar usually found varies 
from 0.5 to 3 per cent.; larger amounts may, however, also be encoun- 
tered, and 1 case is on record in which 8 per cent, was present. 2 

Very common also, as I have indicated, is the digestive glucosuria 
of alcoholics, and there can be little doubt that the habitual ingestion 
of large quantities of beer and spirits is apt in the course of time to lead 
to a more than temporary insufficiency of the carbohydrate metab- 
olism. In the course of his investigations in this direction, Krehl 3 
found among the Jena students that the proportion of those in whose 
urine sugar appeared apparently varied with the different kinds of 
beer, but was much greater after morning drinking. Of 14 who 
drank bock or export beer in the morning, 5 had glucosuria. 
After the evening drinking, amounting in 1 case to seven liters, of 
19 only 1 had sugar in the urine, and with Bavarian beer 1 of 11. 

Of diseases of the skin, digestive glucosuria is notably associated 
with psoriasis; and it is interesting to note that the same disease is 
not infrequently seen in diabetic patients. Gross thus records 5 
cases, in 4 of which the psoriasis had existed for many years 
before the appearance of diabetic symptoms. Similar instances are 

1 "Ueber d. Einfluss d. Schilddriisenbehandl. auf. d. Kohlendydratstoffwechsel/ 
Berlin, klin. Woch., 1897, p. 518. 

2 R. v. Bleiweiss, " Ueber alimentare Glycosurie e saccharo bei acuten, fieber 
aften Infektionskrankheiten," Centralbl. f. inn. Med., 1900, No. 2. 

3 "Alimentare Glycosurie nach Biergenuss," Centralbl. f. inn. Med., 1897, No. 40 



478 THE URINE 

recorded by Strauss, Grube, Polotebuoff, Nielssen, Schiitz, and others. 
Nagelschmidt 1 was able to produce glucosuria by the ingestion of 
100 grams of glucose in 8 cases out of 25. 

During pregnancy digestive glucosuria is also frequently observed, 
and is by some regarded as a fairly constant symptom and of diag- 
nostic importance. The amount is variable, and while Lanz 2 records 
1 case in which 29.6 grams of glucose were found after the inges- 
tion of 100 grams, such figures are certainly uncommon, and as a 
general rule less than 3 grams are recovered from the urine. After 
delivery the power of assimilation for glucose no longer appears to 
be subnormal. The milder form of glucosuria in pregnancy is dur- 
ing the last week or two accompanied by lactosuria. 

A digestive glucosuria has further been observed in acute and 
chronic lead poisoning, poisoning with nitrobenzol, aniline dyes, 
opium, atropine, and carbon monoxide; in the early stages (the first 
twelve days) of acute phosphorus poisoning; in the febrile form of 
embarras gastrique, etc. In these conditions, however, the phenome- 
non has received little attention. 

In patients afflicted with disease of the heart, liver, and kidneys 
Gobbi 3 observed a digestive glucosuria, after the ingestion of from 
100 to 200 grams of glucose, if diuretin was at the same time 
administered. 

Very important is the fact that in diabetes mellitus the sugar may 
at times disappear from the urine, while its elimination is replaced 
by an excessive excretion of uric acid or phosphates. In such cases 
glucosuria may be produced with ease by the ingestion of 100 grams 
of glucose, a point which may be of value in diagnosis. The exhibi- 
tion of such amounts of sugar in true diabetes while glucosuria already 
exists will cause an increased elimination, while this apparently does 
not occur in other forms of glucosuria. Interesting further is the 
fact that in diabetic patients an increased elimination of sugar can be 
produced by the administration of full doses of copaiba. That this 
drug is in itself capable of lowering the limit of the assimilation of 
glucose has recently been shown by Bettmann. A digestive glucosuria 
was thus produced in 4 patients out of 12 of whom copaiba had 
been given for one week in amounts varying from 1 to 2 grams. 

The digestive glucosuria to which reference has been made in the 
preceding pages is generally spoken of as the digestive glucosuria e 
saccharo. Similar results have been obtained after the administra- 
tion of starches in excess, viz., 150 to 200 grams. But while a 
digestive glucosuria e saccharo is regarded only as a t possible indica- 
tion of a pathological alteration of the carbohydrate metabolism, 

1 "Psoriasis und Glycosurie," Berlin, klin. Woch., 1900, No. 2. 

2 Wien. med. Presse, 1895, vol. xxxvi. 

3 "La glucosuria da diuretina," H Policlinico, 1900, No, 5. 



CHEMISTRY OF THE URINE 479 

it is generally thought that every glucosuria ex amylo 1 is indicative 
of a definite disturbance in the sense of diabetes, unless special 
factors, such as an increase of the surrounding temperature, dimin- 
ished radiation of heat, or complete lack of muscular activity, are 
active. Strauss, however, has shown that in cases in which a some- 
what more than temporary predisposition toward glucosuria e sac- 
charo exists, as in alcoholics, for example, a coincident tendency 
toward glucosuria ex amylo may likewise be demonstrated. 4 As a 
result of his experiments he concludes that the difference between 
the digestive glucosuria e saccharo and glucosuria ex amylo is essen- 
tially a question of degree. Ceteris paribus, it appears that harm- 
ful influences of a slight character lead to glucosuria e saccharo, 
while grave insults call forth glucosuria ex amylo. It results prac- 
tically that the prognosis in those cases in which digestive glucosuria 
follows a temporary insult is far better than when the carbohydrate 
metabolism is permanently damaged, and especially when a gluco- 
suria ex amylo accompanies a glusosuria e saccharo. In the first 
instance it is scarcely likely that true diabetes will develop in the 
course of time, while in the latter this is at least possible. 

Aside from the digestive form of glucosuria which has just been 
considered, and which is produced artificially, an idiopathic transi- 
tory form is also known to occur. A transitory glucosuria, appar- 
ently of central origin, is thus noted in connection with lesions 
affecting the central as well as the peripheral nervous system, such 
as tumors and hemorrhages at the base of the brain, lesions of the 
floor of the fourth ventricle, cerebral and spinal meningitis, concus- 
sion of the brain, fracture of the cervical vertebrse, tetanus, sciatica; 
following epileptic, hystero-epileptic, and apoplectic seizures, mental 
shock produced by railroad accidents (traumatic neuroses), etc.; 
mental strain and worry, fatigue, and anxiety. Glucosuria follow- 
ing epileptic and apoplectic attacks, however, does not appear to 
be so common as is generally believed, v. Jaksch was unable to 
demonstrate the presence of sugar in 50 recent cases of hemiplegia, 
and in a large number of cases of epilepsy, with urines voided within 
the first few hours following the seizure, I have reached only negative 
results. 

In Basedow's disease transitory glucosuria may also occur, and 
it is well established that a relation may exist between the disease in 
question and the complex of symptoms designated as diabetes mellitus. 2 

1 E. Kiilz, Beitrage zur Pathol, u. Therap. d. Diabetes, Marburg, 1874, vol. i, 
p. 110. 

2 Dumontpallier, "Goiter exophthalmique et glycosurie," Compt.-rend. de la 
soc. de biol., 1867. O'Neill, " Exophthalmic Goitre and Diabetes occurring in 
the Same Person," Lancet, 1878, pt. i, p. 9. S. Bettmann, Munch, med. Woch., 
1896, vol. xliii, Nos. 49 and 50. E. Grawitz, Fortsch. d. Med., 1897, vol. xv. 
K. Osterwald, Inaug. Diss., Gottingen, 1898. H. Stern, Jour. Amer. Med. Assoc, 
1902, vol. xxxix, p. 972. 



480 THE URINE 

Siegmund noted a transitory glucosuria in 52.38 per cent, of 
general paretics, in 7.4 per cent, of epileptics, and in 3.77 per cent, 
of dementia cases, while it was not observed in other mental dis- 
eases. In reference to the postepileptic glucosuria which has been 
noted by some of the older observers more especially, an analysis of 
their work has led me to the conclusion that their inferences were 
scarcely justifiable, as a wholly satisfactory proof of the presence of 
sugar has not been furnished. 1 

In cases of cholelithiasis, contrary to what has been maintained 
by one or two observers, glucosuria is unusual. 

It is well known that Claude Bernard experimentally produced 
a transitory glucosuria by puncturing a certain spot in the floor 
of the fourth ventricle, the supposed origin of the hepatic vaso- 
motor nerves, and it is not improbable that this neurotic form of 
glucosuria is due to some direct or reflex influence affecting that por- 
tion of the medulla. 

The transitory glucosuria occasionally observed in acute febrile 
diseases, such as typhoid fever, scarlatina, measles, cholera, diph- 
theria, influenza, and especially malaria, particularly during con- 
valescence, may possibly be referable to the action of specific 
toxins upon this centre. Seegen reports 5 cases of malaria 
with " diabetes" in which both conditions disappeared under the 
administration of quinine. In diphtheria glucosuria appears to be 
of common occurrence. Binet thus obtained a positive result in 
29 cases out of 70; 27 times in severe infections out of 38, and 
twice in mild cases out of 32. I have personally found a transi- 
tory glucosuria in 4 cases out of 32; the infection in these was 
of moderate severity. Hibbard and Morrissey arrived at similar 
results. 2 

A glucosuria of toxic origin has been noted in cases of poisoning 
with curare, chloral hydrate, sulphuric acid, arsenic, alcohol, carbon 
monoxide, morphine, etc., and even after simple transfusion of nor- 
mal salt solution into the blood. Phloridzin, a glucoside obtained 
from the bark of the root of the apple tree, will likewise cause sugar 
to appear in the urine. The glucosuria thus produced is, however, 
only temporary, and ceases upon withdrawal of the drug. 3 Of 
interest is the glucosuria which occasionally follows the administra- 
tion of thyroid extract or of iodothyrin, as there is evidence to show 
that in such cases a special predisposition to glucosuria exists. When 
carried to an extreme degree true diabetes may develop, which 
subsequently cannot be arrested by withdrawal of the substance. 4 

1 See, also, Araki., Zeit. f. phys. Chem., vol. xv, p. 363. 

2 "Glycosuria in Diphtheria," Jour. Exper Med., vol. iv, p. 137. 

3 Zuntz, " Zur Kenntniss d. Phloridzindiabetes," Du Bois' Archiv, 1895, p. 570. 

4 H. Strauss, "Neurogene and thyreogene Glucosurie," Deutsch. med. Woch., 
1897, Nos. 19 and 20. 



CHEMISTRY OF THE URINE 481 

The occurrence of a transitory glucosuria under the conditions 
above mentioned, and which may be met with in almost any disease, 
moreover, while interesting from a theoretical standpoint, must in 
the majority of instances be regarded as a medical curiosity only, 
and it is but rarely possible to draw either diagnostic, prognostic, or 
therapeutic conclusions from its existence. 

A persistent form of glucosuria is noted in connection with certain 
lesions of the brain, especially those affecting the floor of the fourth 
ventricle, and is at times of considerable value in diagnosis. This 
is also observed after removal of the thyroid gland, and in cases in 
which thyroid extract has been administered in unduly large amount. 

A continuous elimination of sugar, however, is noted principally 
in the complex of symptoms to which the term diabetes mellitus has 
been applied. 

Diabetes mellitus is essentially a persistent form of glucosuria 
associated with the occurrence of a more or less intense polyuria and 
a greatly increased elimination of all the metabolic products normally 
found in the urine, with the exception of uric acid, which is usually 
present in diminished amount. In the more advanced cases aceto- 
nuria, lipuria, and lipaciduria may also exist. Diabetes, however, is 
not a persistent form of glucosuria in an absolute sense of the word, 
as periods may occur in the course of the disease when glucose is 
temporarily absent. 

The quantity of sugar excreted may be very large, and 180 to 360 
grams pro die are amounts which may be frequently observed. 
This quantity may diminsh to zero under various conditions, such 
as the occurrence of intercurrent diseases, but often also without any 
apparent cause, and not infrequently in the condition which has been 
termed diabetic coma. Cases are also observed in which from begin- 
ning to end mere traces are eliminated, the total amount of sugar 
not exceeding a few grams, while the course of the disease rapidly 
tends toward a fatal termination, so that the severity of the pathological 
process cannot be measured by the amount of sugar eliminated. 

At the same time it should be remembered that diabetes cannot 
be excluded by one or even more negative urinary examinations, and 
the value of repeating such examinations three or four hours after 
the exhibition of 100 grams of glucose, as indicated, cannot be too 
strongly urged. 

Clinicians are in the habit of determining the severity of a case, 
to a certain extent at least, from the condition of the urine under a diet 
free from starches and sugars, and generally regard those cases as the 
more serious in which the glucosuria does not disappear under a diet 
of this character, while a more favorable prognosis is given if the 
sugar disappears. It should be remembered, however, that there 
are numerous exceptions to this rule, and that a light case — i. e., 
one in which the sugar disappears under appropriate dietetic treat- 
31 



482 THE URINE 

ment — may suddenly exhibit symptoms seen only in the most severe 
forms, or succumb to one of the numerous intercurrent maladies, 
while apparently severe cases may assume the more benign type. 

It may not be out of place in this connection to say a few words 
regarding the specific gravity of the urine. While usually very high, 
varying between 1.030 and 1.060, as pointed out in the chapter 
on Specific Gravity, comparatively low figures are noted at times, 
such as 1.012, corresponding to a quantity of urine not exceed- 
ing 1000 c.c, and implying, of course, a diminished elimination 
of solids. This is especially marked in those cases described by 
Hirschfeld, 1 in which, as pointed out in the chapter on Urea, the 
resorption of nitrogenous material from the digestive tract is below 
the normal. Polyuria, a fairly constant symptom of the more com- 
mon types of diabetes mellitus, is much less pronounced in Hirsch- 
feld's form, and may be altogether absent, although it is true that 
this may occur in ordinary diabetes also. 

The simultaneous occurrence of glucosuria, acetonuria, lipuria, 
and lipaciduria (which see) is probably always indicative of true 
diabetes. 

It is, of course, impossible to enter here into a detailed considera- 
tion of the origin of diabetes. Suffice it to say that a persistent glu- 
cosuria, aside from nervous influences, may be referable, on the one 
hand, to an inability on the part of the liver to transform into gly- 
cogen all of the sugar which is carried to this organ; or, on the other 
hand, to an inability on the part of the muscular system of the body 
to utilize all the sugar sent to it. Accordingly, we may distinguish 
between a hepatogenic and a myogenic diabetes. 

Within recent years it has been shown that pancreatic disease is 
frequently associated with diabetes, and while the number of cases 
in which no pancreatic lesions are discovered is still too large to war- 
rant the conclusion that disease of this organ is invariably associated 
with glucosuria, it still must be admitted that lesions of the pancreas 
are the more frequently met with in diabetes the more carefully the 
organ is examined. So much appears to be certain, that diabetes 
may be produced by pancreatic disease. As to the manner, how- 
ever, in which such a result can occur we are in ignorance. In 
this connection it is interesting to note that, according to Opie, dis- 
ease of the areas of Langerhans more especially is associated with 
the clinical picture of diabetes, while lesions affecting the secreting 
portion of the gland only do not influence the carbohydrate metab- 
olism. 2 These observations of Opie have been largely confirmed by 
other observers. 

In cancer of the pancreas glucosuria only occurs in a small per- 

1 " Ueber eine neue klinische Form d. Diabetes," Zeit. f. klin. Med., vol. xix, pp 
294 and 325. 

» Opie, Jour, Exper. Med., 1901,>ol.>,¥p. 527, 



CHEMISTRY OF THE URINE 483 

centage of cases — 3 of 21, as reported by Pearce. 1 In 1 of these 
there was a true cancer diabetes with involvement of the islands, 
while in a second case the glucosuria was intermittent without mani- 
fest changes. 

Hirschfeld pointed out that while in the majority of diabetic patients 
the proteid food is quite satisfactorily utilized, the assimilation of 
fats and albumins is much below normal in others, and particularly 
so in the pancreatic cases. (See also Urea.) Observations in this 
direction are as yet very scanty, so that a definite opinion cannot be 
expressed regarding the utility in diagnosis of investigations similar 
to those of Hirschfeld. 

Whether or not a renal and a thyroigenic diabetes exists, as has 
been suggested, remains an open question. 2 That Basedow's disease 
may be associated with diabetes mellitus I have already pointed out. 

Tests for Sugar. — The tests for sugar usually employed in the 
clinical laboratory depend upon the following properties of sugar: 

1. In the presence of alkalies it acts as a reducing agent upon 
certain metallic oxides, such as those of copper and bismuth (Feh- 
ling's, Trommer's, Bottger's, and Nylander's tests). 

2. In the presence of yeast (Saccharomyces cerevisise) it under- 
goes fermentation, with the formation of ethyl alcohol, carbonic acid, 
succinic acid, glycerin, amyl alcohol, etc. (fermentation test). 

3. With phenylhydrazin sugar forms an insoluble crystalline com- 
pound — phenylglucosazone. 

4. Solutions of glucose turn the plane of polarized light to the 
right, from which property glucose has also received the name 
dextrose. 

In every case the urine should first be tested for the presence of 
albumin, which should be removed by boiling. 

Trommer's Test. 3 — A few cubic centimeters of urine are strongly 
alkalinized with sodium hydrate solution, and treated with a 5 per 
cent, solution of cupric sulphate, added drop by drop, until the 
cupric oxide formed is no longer dissolved. The mixture is care- 
fully heated, when in the presence of sugar a yellow precipitate of 
cuprous hydroxide is formed, which gradually settles to the bottom 
as a sediment of red cuprous oxide. 

It is important to note that while sugar, unless present in mere 
traces, can readily be detected in this manner, other substances are 

1 Amer. Jour. Med. Sci., Sept., 1904, p. 478. 

2 Diabetes: J. Seegen, Die Zuckerbildung im Thierkorper, Berlin, 1890, p. 260. 
v. Noorden, Pathol, d. Stoffwechsels, Berlin, 1893. Seegen, " Ueber d. Zucker- 
gehalt d. Blutes von Diabetikern," Wien. med. Woch., 1886, Nos. 47 and 48. 
F. W. Pavy, " Ueber die Behandlung von Diabetes mellitus," Verhandl. d. X. 
Internat. Med. Congr., 1891, ii, Abt. 5, p. 80. P. F. Richter, " Nierendiabetes," 
Deutsch. med. Woch., 1899, p. 840. 

3 Annal. d. Chem. u. Pharm., 1841, vol. xxxix, p. 361. 



484 THE URINE 

or may be present in the urine, such as uric acid, kreatin and krea- 
tinin, allantoin, nucleoalbumin, milk sugar, pyrocatechin, hydro- 
quinone, and bile pigment, which likewise reduce cupric oxide. Fol- 
lowing the ingestion of benzoic acid, salicylic acid, glycerin, chloral, 
sulphonal, etc., reducing substances also appear. These may 
generally be disregarded, it is true, if care is taken not to boil the 
urine after the addition of the cupric sulphate, as the precipitation 
of cuprous oxide in the presence of sugar takes place before this point 
is reached. Unfortunately, however, the test when thus applied 
yields negative results, or results which are doubtful, if traces only 
are present, so that it cannot be utilized, as a rule, in the study of 
transitory or digestive glucosuria. 

Fehling's Test. 1 — This is a modification of the test just described, 
and can be recommended only with the same restrictions. 

Two solutions are employed, which must be kept in separate 
bottles, the one containing 34.64 grams of crystallized cupric sul- 
phate, dissolved in 500 c.c. of distilled water, and the other 173 
grams of potassium and sodium tartrate and 50 to 60 grams of 
potassium hydrate, dissolved in an equal volume of water. Equal 
parts of the two solutions, mixed in a test-tube and diluted with 
four times as much water, are boiled, when a small amount of urine 
is added. In the presence of sugar a precipitate of the yellow 
hydroxide of copper or of red cuprous oxide will be produced; but 
care should be taken only to warm, and not to boil the solution after 
the addition of the urine. 

Not infrequently it will be observed that upon standing, when no 
precipitation has occurred previously, the blue color of the mixture 
changes to an emerald green, while the solution at the same time 
becomes turbid. Such a phenomenon should not be referred to the 
presence of sugar, as it is in all probability due to the action of other 
reducing substances, such as those mentioned above. 

Boettger's Test with Nylander's Modification. 2 — A few cubic centi- 
meters of urine are treated with Almen's solution in the pro- 
portion of 11 to 1. This is prepared by dissolving 4 grams 
of potassium and sodium tartrate, 2 grams of bismuth subnitrate, 
and 10 grams of sodium hydrate in 90 c.c. of water, heating the 
solution to the boiling point and filtering upon cooling, when it 
should be kept in a colored glass bottle. The mixture of urine 
and Almen's fluid is thoroughly boiled, when in the presence of 
sugar a grayish, dark-brown, and finally a black precipitate, con- 
sisting of bismuthous oxide or of metallic bismuth, is obtained. 
Albumin, if present, must first be removed, as, owing to the sulphur 
contained in the albuminous molecule, alkaline sulphides would be 

1 Annal. d. Chem. u. Pharm., 1849, vol. lxxii. p. 106. 

2 Zeit. f. physiol. Chem., 1883, vol. viii, p. 175, 



CHEMISTRY OF THE URINE 



485 



formed upon boiling, and, acting upon the bismuth, give rise to the 
formation of black bismuth sulphide, which might be mistaken for 
metallic bismuth. Rhubarb pigment, as well as melanin and melan- 
ogen (which see), and free hydrogen sulphide must also be absent, 
as misleading results will otherwise be obtained. 

Nylander's test, as well as that of Trommer and Fehling, is, 
however, not without objections, as a partial reduction of the bis- 
muth subnitrate may be produced by other substances, such as 
kairin, tincture of eucalyptus, turpentine, and large doses of quinine. 

Fermentation Test. 1 — This is based upon the fermentative decom- 
position of sugar with the formation of carbon dioxide and alcohol. 




Fig. 148. — Einhorn's saccharimeter. 

It should be resorted to in all doubtful cases. The test is now almost 
always carried out in special fermentation tubes, such as those of 
Einhorn (Fig. 148) and Lohnstein (Fig. 149). To this end a small 
piece of compressed yeast (a fair sized pill) is broken up in a test- 
tubeful of urine. It is better to do this with a glass rod than by 
shaking. The fermentation tube is filled with this mixture, care 
being taken that no bubbles of air remain at the top. A little mercury 
is poured in, so as to occlude the lower bend, after which the tube is 
kept at a temperature of 30° to 38° C. for twenty to twenty-two hours. 
At the end of this time it is inspected to see whether any gas has 
been formed. In the case of sugar urines it can readily be proven 



E. Salkowski, Berlin klin. Woch., 1905, p. 48. 



486 THE URINE 

that the gas is carbon dioxide by introducing some caustic alkai into 
the tube, when the gas is absorbed. 

In every case it is necessary to make a control test with normal 
urine of approximately the same concentration, as the common com- 
mercial yeast always develops a little carbon dioxide by itself. A 
little bubble is thus usually seen. But the same may occur from the 
liberation of gas which may be present in absorption, when the test 
is kept at the temperature indicated. Unless traces of sugar (less 
than y 1 ^ per cent.) be present no difficulty will result from this fact, as 
the volume of gas in the sugar urine will exceed that of the control. 
But when smaller quantities are present some doubt may arise. In 
that case an attempt must be made to increase the volume of gas by 
heating, when the sugar urine owing to the presence of carbon dioxide 
will show a larger bubble of gas than the control. This may be done 
on a boiling water bath by placing both fermentation tubes in a large 
beaker filled with water such that the tops of the tubes are just 
covered, and heating for half an hour. 

If a positive result is obtained with the fermentation test the pres- 
ence of a fermentable sugar is proven; the question whether this is 
dextrose or levulose, which alone enter into consideration in disease, 
is practically unimportant. Should blood, pus, albumin, or albumose 
be present, these should first be removed. 

Rarely it will happen that the urine undergoes ammoniacal decom- 
position in the tubes; if it does occur the examination should be 
repeated. 

Phenylhydrazin Test. 1 — As originally proposed by v. Jaksch, the 
test is conducted as follows: 6 to 8 c.c. of urine are treated with 
0.4 to 0.5 gram of phenylhydrazin hydrochlorate and 1 gram of 
sodium acetate, and warmed until the salts have been dissolved, a 
little water being added if necessary. The tube is placed in boiling 
water for twenty to thirty minutes, and then transferred to a beaker 
filled with cold water. If sugar is present in moderate amounts, a 
bright-yellow, crystalline deposit will at once be thrown down and 
partly adhere to the sides of the tube. But even in the presence of 
mere traces a careful microscopic examination will reveal the 
presence of crystals of phenylglucosazone. These are seen singly or 
arranged in bundles and sheaves composed of delicate, bright-yellow 
needles which are insoluble in water. 

Still more convenient is the following modification of the test, as 
suggested by Cipollina: 2 5 drops of pure phenylhydrazin, 0.5 c.c. 
of glacial acetic acid, or 1 c.c. of 50 per cent, acetic acid are placed 
in a test-tube together with 4 c.c. of urine. The mixture is boiled 
for about one minute over a small flame, while shaking so as to avoid 

1 v. Jaksch, Zeit. f. klin. Med., 1886, vol. xi, p. 20. 

2 Deutsch. med. Woch., 1901, vol. xxvii, p. 334. 



CHEMISTRY OF THE URINE 487 

bumping as much as possible; 4 or 5 drops of sodium hydrate solution 
(specific gravity 1.16) are added, but the solution must remain acid; 
the boiling is continued for a few seconds and the mixture then 
allowed to cool. The rapidity with which the glucosazone crystals 
separate out depends somewhat upon the specific gravity of the urine. 
If this is low they form in a few minutes, even though the amount 
of sugar does not exceed 0.05 per cent. If, on the other hand, the 
specific gravity is high, yellow balls and thornapple forms* result, 
while typical rosettes develop only after twenty to thirty minutes, and 
at times one is even then left in doubt as to the result. If the urine 
contains more than 0.2 per cent, of sugar, however, even though the 
specific gravity be high, the formation of typical crystals occurs within 
a few minutes. If with this modification no crystals are obtained at 
the expiration of an hour, we may infer that no sugar is present. 

This test, properly applied, is undoubtedly not only the most deli- 
cate, but at the same time the most reliable, as no other substances 
which may be present in the urine, excepting maltose and certain 
pentoses, will give rise to the formation of an osazone. Hence, when- 
ever doubt is felt as to the nature of a substance reacting in a posi- 
tive manner with the reagents described above, recourse should be 
had to this test. It has been stated that maltose forms an exception; 
this, however, will never become embarrassing, as the microscopic 
appearance of the maltosazone crystals differs from that of the phenyl- 
glucosazone. The melting point of phenylglucosazone (205° C), 
moreover, is about 15° C. higher than that of the maltosazone — 
190° to 191° C. To determine this point, it is necessary to filter 
off the osazone, and, after washing with water, to dissolve it upon a 
filter by means of a little hot alcohol. From this alcoholic solution 
it is reprecipitated by water, when it may be collected and dried over 
sulphuric acid. The melting point is then determined according to 
the usual methods. 

The pentosazones also can be readily distinguished from glucosazone 
by their melting points (which see). 

The amount of lactose which may be found in the urine is far too 
small to give rise to the formation of an osazone when the test is 
directly applied to the urine. 

With the conjugate glucuronates phenylhydrazin also combines to 
form crystalline compounds, but these may likewise be distinguished 
by their melting points and the form of the crystals. Such com- 
pounds, moreover, are usually not present in amounts sufficient to 
give rise to confusion. (See Glucuronic Acid.) 

Polarimetric Test. — Glucose turns the plane of polarized light 
to the right, but the same may be said of maltose, the degree of 
polarization of which is even more marked, so that it may be impos- 
sible to state in a given case whether such rotation is referable to a 
large quantity of glucose or to a smaller quantity of maltose. The 



488 



THE URINE 



latter substance, however, occurs in the urine but rarely, and may be 
recognized not only by the microscopic appearance of its osazone, 
but also by the fact that its power of reduction is increased in the 
presence of sulphuric acid and by the application of heat. 

An error which may further arise with the employment of the 
polarimetric method is referable to the fact that if glucose is pres- 
ent in only small amounts, while the urine contains large quantities 
of /5-oxybutyric acid, the latter turning the plane of polarized light 
to the left, it may happen that the rotation in this direction will neu- 
tralize or even counterbalance any rotation to the right, which may 
be due to glucose. In such cases, however, the urine will react in a 
positive manner with the other reagents described, and the fermented 
urine will, moreover, turn the plane of polarization still more strongly 
to the left, indicating the presence of a dextrorotatory substance, and 
in all probability of glucose. 

The delicacy of this method varies with the instrument employed; 
the figures given below were obtained with the apparatus of Lippich, 
which yields the best results. (For a description of this method see 
the Quantitative Estimation of Sugar by Means of the Polarimeter.) 

Table showing the Delicacy of the Tests described. 

Trommer's test . 0025 per cent. 

Fehling's test 0.0008 

Nylander's test 0.025 

Fermentation test . . . 1-0 . 05 

Phenylhydrazin test 0.025-0.05 

Polarimetric test 0.025-0.05 



Table 


SHOWING THE BEHAVIOR OF THE VARIOUS FORMS OF 


Sugar which 




MAY OCCUR IN THE URINE 


TOWARD THE TESTS DESCRIBED. 




Trommer's, viz., 
Fehling's test. 


Nylander's 
test. 


Fermenta- 
tion test. 


Phenylhydrazin 
test. 


Polarimetric 
test. 


Glucose. 


Positive reaction. 


Positive 
reaction. 


Positive 
reaction. 


Positive reaction; 
melting point 
205° C. 


Rotation toward 
the right. 


Levulose. 


Positive reaction. 


Positive 
reaction. 


Positive 
reaction. 


Same osazone ob- 
tained as with 
glucose, only 
more rapidly. 


Rotation toward 
the left. 


Maltose. 


Positive reaction. 


Positive 
reaction. 


Positive 
reaction. 


A maltosazone is 
formed; melting 
point 190°-191° 
C. 


Rotation toward 
the right. 


Lactose. 


Positive reaction. 


Positive 
reaction. 


No reaction 
or only a 
very faint 
one. 


No reaction in the 
concentration in 
which it may oc- 
cur in the urine; 
melting point 
200° C. 


Rotation toward 
the right; in- 
creased by boil- 
ing with a 2. 5 per 
cent, solution of 
sulphuric acid. 


Laiosel 


Positive reaction 
on boiling only: 
1.2-1.8 per cent, 
more is obtain- 
ed than by the 
polarimeter. 


Positive 
reaction. 


No reaction. 


With phenylhy- 
drazin a yellow- 
ish-brown, non- 
crystallizable oil 
is obtained. 


No reaction, or 
rotation toward 
the left. 



CHEMISTRY OF THE URINE 489 

Clinically, it is unimportant to search for minute traces of sugar, 
such as may be found in every normal urine, and the reader is referred 
to special works on physiological chemistry for a consideration 
of the methods generally employed. (See method of Baumann and 
v. Udranszky.) 

Quantitative Estimation of Sugar. — The methods used in the 
quantitative estimation of sugar are essentially based upon the 
qualitative tests described. * 

Fehling's Method. — The Fehling solution (see above: qualitative 
tests) must be accurately standardized as follows: 0.2375 gram of 
pure crystallized cane sugar, dried at 100° C, is dissolved in 40 c.c. 
of distilled water, to which 22 drops of a 10 per cent, solution of sul- 
phuric acid have been added. This solution is kept on the boiling 
water bath for an hour, when it is allowed to cool and diluted to 
100 c.c. with distilled water; 20 c.c. of this solution will then con- 
tain exactly 0.05 gram of glucose, corresponding to 10 c.c. of 
Fehling's solution, if this is of the required strength. If too strong, 
so that 21 c.c. of the sugar solution, for example, are required to 
obtain a complete reduction of the copper, the strength of Fehling's 
solution may be determined according to the equation: 20: 0.05: : 
21: x; and x= 0.0525. If too weak, on the other hand, so that 19 
c.c, for example, are required, its strength is similarly determined: 
20: 0.05: : 19: x; and ic=0.0475. 

If the solution is of the theoretically required strength 10 c.c. will 
correspond to 0.05 gram of glucose. 

If then urine is added to this quantity until complete reduction 
has taken place, the amount of sugar in a given specimen of urine 
can be calculated according to the following equation: 

y : 0.05 : : 100 : x; and x =—, 

y 

in which y indicates the number of cubic centimeters of urine required 
to reduce the 10 c.c. of Fehling's solution, and x the amount of sugar 
contained in 100 c.c. of urine. 

As the best results are obtained if from 5 to 10 c.c. of urine are 
used in one titration, it is often necessary to dilute the urine to this 
end; in the determination of this point the specific gravity may serve 
as a guide. As a general rule, urines of a specific gravity of 1.030 
should be diluted five times, and if the density is still higher ten 
times. Albumin, if present, must first be removed by boiling: 10 
c.c. of Fehling's solution diluted with 40 c.c. of water are placed 
in a porcelain dish and boiled. While boiling, the diluted urine 
is added from a burette, 0.5 c.c. at a time, when, as a rule, the 
precipitated cuprous oxide will settle, so that the white sides of the dish 
may be seen through the blue field. In my experience it is very helpful 
to boil the mixture for a few moments after every addition of urine and 



490 THE URINE 

to stir thoroughly each time with a rubber-tipped rod. In this way 
the precipitate is prevented from forming a coating on the vessel and 
settles down more readily. As the end point is reached every trace 
of blue has disappeared and the liquid has a faint yellowish tinge 
owing to beginning caramelization of the excess of sugar by the caustic 
alkali. 

If any doubt should arise whether the end point has been reached, 
tiny droplets of the mixture should be placed upon ferrocyanide paper 
(prepared by soaking filter paper in a moderately dilute solution of 
potassium ferrocyanide) . If unreduced copper is still present a brown 
color results. The result is regarded as positive only, if the brown 
develops at once. If it occurs only after several seconds the final 
point has been reached or passed. 

Prolonged boiling always brings some copper into solution again. 
It is hence advisable to make two examinations always, the one 
approximately only, and the second as the final one. 

The calculation is then made as indicated above. 

Example. — The volume of urine for twenty-four hours was 4000 
c.c. It was diluted five times; 6 c.c. of the diluted urine brought 
about the complete reduction of 10 c.c. of Fehling's solution; the 6 
c.c. hence contained 0.05 gram of sugar; 100 c.c, accordingly, con- 
tained 0.833 gram. As the urine had been diluted five times this 
figure must be multiplied by 5 = 4.165, which is the percentage for the 
native urine. The amount for the twenty-four hours was hence 
4.165 = 40 (hundreds) X 166.6 grams. 

Gerrard and Allan's Method (Modified by Rudisch and Celler). 1 
— To obviate some of the difficulties which attach to Fehling's 
method Rudisch and Celler have recently suggested the following 
modification of Gerrard and Allan's method: 

"To four parts by volume of a 50 per cent, solution of potassium 
sulphocyanate, chemically pure, is added one part by volume of a 
mixture of equal parts of Fehling's copper sulphate and alkaline 
solutions. 25 c.c. of this solution are placed in a porcelain dish, 
and the urine to be tested added drop by drop from a burette until 
the blue color of the copper entirely disappears. Throughout the 
titration the solution should be slowly boiled and constantly stirred 
with a glass rod. The end reaction is extremely sharp, the fluid 
becoming colorless or assuming a faint-yellow tinge. The advantages 
of this method are: (1) only one titration is necessary, as potassium 
sulphocyanate does not decolorize the copper solution; (2) potassium 
sulphocyanate is not poisonous; (3) as the mixture is stable a con- 
siderable quantity may be made to be kept as ' stock/ Such a 
'stock' solution was found to be unaffected after four months' 
exposure to heat and sunlight. 

1 Jour. Amer. Med. Assoc, January 26, 1907. 



CHEMISTRY OF THE URINE 491 

" With aqueous solutions of glucose ranging from 0.25 to 6 per 
cent, the results obtained with this method and with the polari- 
scope are identical. With diabetic urines, however, variations of 
from 0.03 to 0.25 per cent, are occasionally found — differences that 
are too small to be of clinical significance. These variations are 
explicable on two grounds. First, substances other than glucose 
(creatinin, uric acid, glucuronic acid) reduce copper and give too 
high a reading with Fehling's solution; secondly, levorotating sub- 
stances (albumin, levulose, /9-oxybutyric acid) may coexist with the 
glucose in the urine, giving too low a percentage with the polariscope. 
To estimate properly the quantity of dextrose in any given specimen, 
therefore, it is necessary to make determinations both with the copper 
solution and with the polariscope. Should the former indicate a 
higher percentage than the latter, levulose should be suspected and 
tested for with the Seliwanoff resorcin-hydrochloric acid method. 
In the absence of levulose the most probable disturbing factor is 
/9-oxybutyric acid, as albumin and other levorotators are precipitated 
when the urine is cleared with lead acetate for the polariscope. 

"Although with undiluted urines containing large amounts of 
dextrose satisfactory results have been obtained with this method, 
the extreme care necessary in titrating under these conditions makes 
it advisable to dilute such urine from five to ten times. It is preferable 
to examine specimens when fresh, but, should it become necessary to 
employ preservatives, toluol, salicylic acid, or carbolic acid may be 
added in small quantities without markedly interfering with the reac- 
tion. Chloroform, on the other hand, must be avoided, as even in 
minute traces its presence vitiates the test. 

" In calculating the percentage of sugar by the above method it 
must be remembered that the titre of the copper solution is unchanged 
by the addition of the solution of potassium sulphocyanate, and that 
the mixture represents Fehling's solution diluted five times. Each 
c.c. of the reagent will therefore be reduced by 1 mgm. of sugar. 

" For example, if for the decolorization of 25 c.c. of the mixture, 
equivalent to 25 mgm. of sugar, 1.2 c.c. of undiluted urine are used, 
then 1 c.c. of the urine will decolorize 25 divided by 1.2=20.8 c.c. of 
the reagent, equivalent to 28.8 mgm. of sugar, or 2.08 per cent. 

"If 0.75 c.c. of urine decolorize 25 c.c. of the reagent, 1 c.c. will 
decolorize 25 divided by 0.75=33.3 c.c. of reagent, equivalent to 33.3 
mgm. of sugar, or 3.33 per cent." 

Differential Density Method. 1 — This method is very useful in 
clinical work, and should be preferred to the more uncertain titra- 
tion with Fehling's solution. 

The specific gravity is accurately ascertained by means of a 

1 Roberts, Lancet, 1862, i, p. 21. Worm-Muller, Pfliiger's Archiv, 1884, vol. 
xxxiii, p. 211, and 1885, vol. xxxvii, p. 479. 



492 THE URINE 

pyknometer, or a hydrometer graduated to the fourth decimal and pro- 
vided with a thermometer indicating tenths of a degree. The tempera- 
ture at which the specific gravity is taken should be that for which the 
hydrometer has been constructed, the urine being heated or cooled 
to the desired degree; 100 to 200 c.c. are set aside in a flask, 
loosely stoppered after the addition of a small piece of yeast, which 
should be finely divided. After twenty-four hours if but little sugar 
is present, or forty-eight hours if there is much, the specific gravity 
is again determined under the precautions given, after having filtered 
the urine. The difference in the specific gravity is multiplied by 230, 
an empirical factor which has been found by dividing the amount of 
sugar ascertained by titration or polarization with the difference in 
the density of the urine after fermentation. The result indicates the 
percentage of sugar. Evaporation must be guarded against by using 
a bulbed safety tube containing some alkaline solution. 

The process may be hastened if to each 100 c.c. of urine 2 grams 
of potassium and sodium tartrate and 2 grams of diacid-sodium phos- 
phate are added, with 10 grams of compressed yeast, and the mixture 
is kept at a temperature of from 30° to 34° C. If but little sugar is 
present, two or three hours will be sufficient. That portion of the 
urine of which the specific gravity is determined before fermentation 
should really be treated in the same manner. It will suffice, however, 
to add 0.022 to the specific gravity found, to make up for the increase 
that would otherwise be observed in the second specimen owing to 
the addition of the salts. 

In every case the urine must be perfectly fresh, as fermenta- 
tion generally begins spontaneously, even after standing a short 
time. 

Einhorn's Method. — This will answer very well for ordinary 
purposes. Two especially constructed and graduated saccharimetric 
tubes (see Fig. 148) are used, one of which is filled with a mixture of 
the suspected urine and yeast, and the other with normal urine and 
yeast, as a control. The examination in general is conducted as 
described before. (See Qualitative Tests for Sugar.) 

Lohnstein's Method. — A very convenient modification of Ein- 
horn's instrument, and one furnishing more accurate results, has been 
introduced by Lohnstein. 1 As will be seen from the accompanying 
figure (Fig. 149), this is essentially a U-tube open at both ends. The 
longer limb is closed during the process of fermentation by a ground- 
glass stopper. This stopper is provided with an air-hole, to which a 
similar hole corresponds in the drawn-out portion of the tube. The 
apparatus is filled with the urine to be examined, through the bulb A, 
while the two air-holes at B are in communication. Care should be 
had that the liquid stands exactly at the mark 0. The stopper is 

1 " Ein neues Gahrungssaccharometer," Berlin, klin. Woch., 1898, p. 866. 



CHEMISTR Y OF THE URINE 



493 



then turned so that all communication between the air and the urine 
is cut off. A little mercury is finally poured into the saccharimeter, 
when the instrument is maintained at a temperature of about 30° to 
38° C. After twelve hours the percentage of sugar is read off directly. 

Precautions : 1 . As every urine contains 
traces of free carbon dioxide, it is well to 
remove this by boiling if we have reason to 
suppose that only a small amount of sugar 
is present. Before adding the yeast the 
urine is, of course, cooled to the surround- 
ing temperature. 

2. As the instrument yields satisfactory 
results only if the urine contains less than 
1 per cent, of sugar, it is necessary to 
dilute it with water when more is present. 
The specific gravity may here serve as an 
index; urines of a specific gravity up to 
1.018 are examined directly; from 1.018 to 
1.022 they are diluted twice, from 1.022 
to 1.028 five times, and those above 1.028 
ten times. 

3. A test-tube, provided with the neces 
sary marks to indicate the degree of dilu- 
tion of the urine, accompanies the instru- 
ment. In every case a globule of yeast, 
approximately 6 to 8 mm. in diameter, is 
added to the urine and shaken in the tube BS FlG - 149.— Lohnstein's s ac - 

charimeter. 





Fig, 150. — Soleil-Ventzke's saccharimeter. 



494 THE URINE 

until an even suspension has been reached. 1 (See also Qualitative 
Tests for Sugar.) 

Polarimetric Method. — For this purpose the saccharimeter of 
Soleil-Ventzke is very convenient (Fig. 150). This consists essen- 
tially of a Nicol prism, A, which may be rotated about the axis of 
the apparatus; a second Nicol prism, at D; vertically placed com- 
pensating prisms, consisting of dextrorotatory quartz, at E, which 
may be moved horizontally by means of a rack-and-pinion adjust- 
ment, turned by a milled head at K, so that light can pass through a 
thicker or thinner layer of the dextrorotatory quartz. At F is a plate 
of levorotatory quartz cut perpendicularly to the optical axis, and 
covering the entire field of vision; at H biquartz plates of Soleil, and 
at I an Iceland-spar crystal ; B and C represent a small telescope, by 
means of which the biquartz plates can be accurately focussed. 
When the compensation prisms of this apparatus are in a certain 
position the levorotation of the plate F will be exactly compensated, 
and the two halves of the field of vision present the same color, while 
the zero of the scale X will coincide with the zero of the vernier Y, 
arranged on the upper surface of the compensators. Any change in 
this position produced by turning the screw K will cause the appear- 
ance of a different color in each half of the field of vision. If now, with 
a zero position, an optically active dextrorotatory or levorotatory sub- 
stance is interposed, the color of each half of the field of vision will 
become altered, but may be equalized again by changing the position 
of the compensators, the degree of change necessary to produce this 
result constituting an index of the power of rotation of the solution 
interposed in the tube M. 

Soleil- Ventzke's apparatus is constructed in such a manner that 
if a solution of glucose is employed, the length of the tube M being 
10 cm., every entire line of division on the scale will indicate 1 per 
cent, of sugar. 

The tube of the saccharimeter should be carefully washed out with 
distilled water, and at least once or twice with the filtered urine, 
when it is placed on end upon a flat surface and filled with the urine, 
so that this forms a convex cup at the end. The glass plate is now 
carefully adjusted, so as to guard against the admission of bubbles 
of air. The metallic cap is placed in position, care being taken to 
avoid undue pressure. The examinations are made in a dark room; 
an ordinary lamp is used, and several readings are taken, until the 
differences do not amount to more than 0.1 or 0.2 per cent. The 
tubes should be thoroughly cleansed immediately after the experiment. 

In every case the filtered urine should be free from albumin, and, 
if markedly colored, should be previously treated with neutral lead 
acetate in substance and filtered. 

1 Lohnstein's saccharimeter may be procured from R. Kaltmeyer & Co., Oran- 
jenburger Str. 45, Berlin. t 



CHEMISTRY OF THE URINE 495 

If it is only desired to demonstrate the presence of sugar, the 
compensators are first brought to the zero position. If now, upon 
interposition of the tube filled with urine, a difference in the color 
of the two halves of the field of vision is noted, the presence of an 
optically active substance in the urine may be assumed; and if the 
deviation is at the same time to the right, the presence of glucose is 
rendered highly probable, while a deviation to the left will generally 
be referable to levulose or /3-oxybutyric acid. Indican, peptones 
(albumoses), cholesterin, and certain alkaloids, it is true, also turn 
the plane of polarization to the left; but as a rule these substances 
need not be considered, as cholesterin occurs but rarely, and indican 
is usually present in only small amounts in diabetic urines. Albu- 
moses, if present, must first be removed. Lactose and maltose, 
which also turn the plane of polarization to the right, may be dis- 
tinguished from each other and from glucose by the phenylhydrazin 
test. Levulose turns the plane of polarization to the left. Oxy- 
butyric acid is practically always associated with the presence of 
glucose, and may be recognized by allowing the urine to undergo 
fermentation, when the filtered urine will become distinctly levo- 
rotatory. 

Lactose. — Lactose 1 is a normal constituent of the urine during the 
last weeks of pregnancy and the first weeks following childbirth. 
The antepartum lactosuria usually amounts to about 1 gram pro 
liter, but may reach 2 grams and rarely even higher figures. The 
postpartum lactosuria is more marked. It reaches its maximum 
between the third and fifth day after labor, the amount varying be- 
tween 1 and 8 grams pro liter. 

After lactation is once well established lactose is not usually found 
in the urine, but it may occur if for any reason milk stasis occurs. 

Occasionally lactosuria is accompanied by a mild grade of gluco- 
suria. 

An alimentary lactosuria may follow the ingestion of 60 grams of 
lactose, though as a general rule 120 grams may be regarded as the 
limit of tolerance. 

The presence of lactose may be inferred if a positive result is 
obtained with Trommer's and Nylander's tests, while the phenyl- 
hydrazin and fermentation tests give negative results. An osazone 
may, however, be obtained from the isolated substance. 

Levulose. 2 — An alimentary levulosuria occurs after the ingestion of 
more than 140 to 160 grams of sugar. In severe cases of diabetes 

1 De Sinetv, Maly's Jahresber., 1874, vol. iii, p. 134. Hempel, Arch. f. Gynak., 
1875, vol. viii, p. 312. Ney, ibid., 1889, vol. xxxv, p. 239. F. Hofmeister, 
"Ueber Laktosurie," Zeit. f. physiol. Chem., 1877, vol. i, p. 101 (lit.). F. A 
Lemaire, ibid., 1896, vol. xxi, p. 442. Commandeur and Porcher, Arch. gen. de 
med., 1904, pp. 2241 to 2305. 

2 Seegen, Centralbl. f. d. med. Wiss., 1884, vol. xxii, p 753. H. Rosin and L, 
Labaud, Zeit. f. klin. Med., vol. xlvii, Heft 1 u. 2. 



496 THE URINE 

levulose may be found in the urine together with glucose, even though 
the food contains neither levulose nor other carbohydrates. Such 
an occurrence is regarded as a grave omen. 

Spontaneous levulosuria unaccompanied by glucosuria has also 
been described. Such urines show a deviation to the left or none at 
all, while the other tests for sugar indicate the presence of a reducing 
substance. 

Maltose. — Maltose, together with glucose, was first found in the 
urine of a patient supposedly the subject of pancreatic disease, asso- 
ciated with an acholic condition of the stools. Since that time it 
has been repeatedly observed in diabetic patients. In one case the 
amount was 27.8 grams pro liter. Similar results have been obtained 
in dogs after extirpation of the pancreas. 1 Its recognition is prac- 
tically dependent upon the formation of its osazone and a deter- 
mination of the melting point of the latter. Such urines, moreover, 
show a larger percentage of sugar on polarization than on titration 
with Fehling's solution. At the same time it will be observed that 
on heating for two hours with hydrochloric acid at 106° F. the polari- 
metric .values become smaller, while the titration values increase. 

Dextrin. 2 — In one case of diabetes dextrin appeared to take the 
place of glucose. It may be recognized by the fact that upon the 
application of Fehling's test the blue liquid first becomes green, 
than yellow, and sometimes dark brown. Traces of dextrin are 
probably present in every urine, but cannot be demonstrated with 
the common tests. 

Laiose. 3 — Laiose has been found in the urine of a diabetic patient. 
It is essentially characterized by the fact that on titration with Feh- 
ling's solution from 1.2 to 1.8 per cent, more sugar is indicated than 
by the polarimetric method. 

Pentoses. — Traces of pentoses probably occur in every urine, but 
are not demonstrable by the common tests. Somewhat larger amounts 
may be found after the ingestion of fruit which is rich in pentoses, 
such as huckleberries, plums, cherries etc. (alimentary pentosuria). 
The tolerance of pentoses normally is less than 30 to 50 grams. If 
such amounts are taken one-half usually reappears in the urine. 

Marked pentosuria has been described in a morphine habitue 
by Salkowski and Jastrowitz, where it alternated with glucosuria. 
Similar cases have been reported by Real, Kulz, and Vogel, and others 
have observed pentosuria in diabetes. Several cases have been 
described in apparently normal individuals and of late a family ten- 
dency to pentosuria has been observed in some cases. In these idio- 
pathic cases arabinose is found, while xylose and rhamnose are met 
with in the alimentary type of the anomaly. 

1 Lepine and Boulud, Compt.-rend., vol. cxxxii, p. 610. 

2 Reichard, Maly's Jahresber., 1876, vol. v, p. 60, 

3 Leo, Virchow's Archiv, vol. cvii. 



CHEMISTRY OF THE URINE 497 

Pentose urines reduce Fehling's solution and Nylander's solution, 
and give rise to the formation of an osazone when treated with phenyl- 
hydrazin. The osazone can be distinguished from that obtained from 
glucose, maltose, or lactose, etc., by the melting point (159° to 160° 
C.). The fermentation test is negative. Xylose and rhamnose turn 
the plane of polarization to the right, while arabinose is optically 
inactive. The presence of pentoses can be definitely established 
with the orcin test. ♦ 

Orcin Test (Bial's Modification 1 of Tollens' Test).— The reagent 
consists of 1 gram of orcin and 25 drops of the liquor ferri chloridi in 
500 c.c. of a 30 per cent, solution of hydrochloric acid. A few c.c. 
of this are heated to boiling in a test-tube and treated with a few drops 
of urine. A green color develops in the presence of pentoses. The 
green pigment can be extracted with amyl alcohol, and on spectro- 
scopic examination it gives rise to a well-defined band of absorption 
in the red portion of the spectrum near the yellow border. 

Tollens' Phloroglucin Test, in which phloroglucin is substituted for 
the orcin, and in which a deep-red color is obtained in the presence 
of a pentose, may also be used, but the reagent indicates the presence 
of glucuronates as well. 

Literature. — E. Salkowski u. M. Jastrowitz, "Ueber eine bisher nicht beo- 
bachtete Zuckerart im Ham," Centralbl. f. d. med. Wiss., 1892, No. . 19. E. 
Salkowski, " Ueber d. Pentosurie," Berlin, klin. Woch., 1895. No. 17. F. Blumen- 
thal, ibid., No. 26; and Zeit. f. klin. Med., vol. xxxvii, p. 415. E. Kiilz u. J. 
Vogel, Zeit. f. Biol., N. F., 1896, vol. xiv, p. 189. E. Salkowski, " Ueber d. 
Vorkommen von Pentosen im Harn," Zeit. f physiol. Chem., 1899, vol. xxvii, 
p. 587. Bial, Ueber Pentosurie, Zeit. f. klin. Med., 1900, vol. xxxix, p. 472. 
Bendix, Munch, med. Woch., 1903, No. 36. Bial, Berlin, klin. Woch., 1904, 
p. 552. 

Glucuronic Acid. 

Glucuronic acid is derived from glucose, and constitutes an inter- 
mediary product of the normal metabolism of the body. In the 
urine it is found only in combination with certain fatty and aromatic 
alcohols, forming compounds which are related to the glucosides 
and are generally spoken of as the conjugate glucuronates. Such 
bodies have been observed in the urine following the ingestion of 
chloral, camphor, naphtol, oil of turpentine, menthol, phenol, mor- 
phine, antipyrine, etc., and traces may also be obtained from nor- 
mal urines. The normal glucuronates are undoubtedly compounds 
of glucuronic acid with phenol, paracresol, indoxyl, and skatoxyl. 
Their amount is exceedingly small, as the greater portion of these 
bodies is normally eliminated in combination with sulphuric acid. 
According to P. Mayer, an increased elimination of glucuronates 

1 Deutsch. med. Woch., 1903, No. 27. 
32 



498 THE URINE 

precedes alimentary glucosuria. Both conditions frequently coexist 
in diabetic individuals. 

Of the quantitative variations of the normal glucuronates and 
their relation to disease, next to nothing is known. Their clinical 
interest centres in the fact that certain glucuronates are capable of 
reducing copper and bismuth in alkaline solution. The glucuronates 
are readily decomposed by boiling with 1 per cent. H 2 S0 4 (for one to 
five minutes). Unless this is previously done reduction of the alka- 
line copper sulphate solution only takes place slowly on prolonged 
heating. But if the cleavage is first accomplished it occurs at once. 
Such urines do not undergo fermentation. The glucuronates turn the 
plane of polarization to the left, while glucuronic acid itself is dextro- 
rotatory. Like the pentoses, the glucuronates give a positive reaction 
with phloroglucin, while they do not react, with orcin (see above). 
With the free acid phenylhydrazin forms crystalline compounds. 

A quantitative method has recently been published by Neuberg and 
Neumann. 1 

Literature. — H. Thierfelder, "Ueber d. Bildung v. Gtykuronsaure," etc., 
Zeit. f. physiol. Chem., 1886, vol. x, p. 163; "Untersuchungen iiber d. Glykuron- 
saure," ibid., 1887, vol. xi, p. 388. P. Mayer, "Ueber d. Ausscheidung u. d. 
Nachweis d. Glykuronsaure," Berlin, klin. Woch., 1899, pp. 591 and 617. P. 
Mayer u. C. Neuberg, Zeit. f physiol. Chem., 1900, vol. xxix, p. 256 



Inosit. 

According to Hoppe-Seyler, traces of inosit may be found in 
the urine under normal conditions. Somewhat larger quantities 
are eliminated following the ingestion of large amounts of water, 
and for this reason possibly inosituria is notably observed in cases 
of diabetes insipidus, in diabetes mellitus, and in chronic intersti- 
tial nephritis. Its occurrence in these diseases is, however, not 
constant. The substance is devoid of clinical interest. It is not a 
carbohydrate, but belongs to the aromatic series, and is commonly 
regarded as hexahydroxybenzol. Its formula is C 6 H 12 6 +H 2 0. 
For methods of isolating the substance from the urine, the reader is 
referred to special works. 2 



Urinary Pigments and Chromogens. 

Under normal conditions urochrome and uroerythrin, to which 
latter the red color of urate sediments is due, .are the only pigments 
which occur preformed in the urine. In disease, on the other hand, 

1 Zeit. f. physiol. Chem., 1905, vol. xliv, p. 127. 

? C. E. Simon, Physiological Chemistry, Lea Bros. & Co. 



CHEMISTRY OF THE URINE 499 

various other pigments may be found, which occur either free or 
in the form of chromogens. Among the former may be mentioned 
hemoglobin, methemoglobin, hematin, hematoporphyrin, urorubro- 
hematin, urofuscohematin, urobilin, the biliary pigments, and melanin ; 
while abnormal chromogens are met with following the ingestion of 
certain drugs, such as santonin, senna, rheum, iodine, etc., as also in 
cases of poisoning with carbolic acid, creosote, etc. The occurrence 
of some of these substances, such as the various forms of blood pig- 
ment, the biliary pigments, and indigo, viz., indican, is of considerable 
clinical interest, while others again are of only minor importance. 

Normal Pigments. Urochrome. — To the presence of this pig- 
ment, which appears to be identical with the normal urobilin of 
MacMunn, but which should not be confounded with the pathological 
urobilin of Jaffe, the normal yellow color of the urine is probably 
largely due. It is supposedly derived from bilirubin, which in turn is 
referable to hematin, and thus from the hemoglobin of the blood. 

In order to obtain urochrome from normal urine, this is acidulated 
with 1 to 2 grams of dilute sulphuric acid pro liter, filtered, and 
saturated with ammonium sulphate in substance, when the flakes 
which are formed, if an excess of the salt has been added, are dried 
and treated with warm, slightly ammoniacal absolute alcohol; the 
pigment is then obtained upon evaporation of the alcohol. 

An alcoholic solution of urochrome, like the urobilin of Jaffe, 
exhibits a beautiful greenish fluorescence when treated with ammonia 
and a few drops of a solution of zinc chloride; but, unlike the latter 
substance, its acidulated alcoholic solutions present a broad band of 
absorption at F, which extends more to the left than to the right of 
this line, while the remainder of the spectrum is at the same time 
absorbed to the right end, from a point somewhat to the left of G. 
Garrod, on the other hand, states that by acting upon urochrome with 
acids he did not succeed in obtaining any product showing the urobilin 
band or yielding the well-known fluorescence with zinc chloride and 
ammonia. But a substance having both these properties was readily ob- 
tained by the action of aldehyde upon an alcoholic solution of the pig- 
ment. In a short time — shorter still when the liquid is warmed — an 
absorption band appears like that of urobilin, and the tint of the solu- 
tion deepens to a rich orange-yellow. With zinc chloride and ammonia 
a brilliant green fluorescence appears, and the band is shifted toward 
the red, as that of urobilin is under like circumstances. The process 
can be stopped at this point by the simple addition of water, for 
aldehyde has no such action upon aqueous solutions of urochrome. 
If, however, the action be allowed to continue, a further change 
ensues; the liquid reddens, and a second band appears in the violet. 
The fluorescence can still be obtained with zinc chloride and am- 
monia, and both bands are shifted toward the red and are closer 
together than before. The reaction with aldehyde, according to 



500 THE URINE 

Garrod, affords a very delicate test for the presence of urochrome 
in alcoholic solutions. The product of the earlier stage, although 
it is not identical with urobilin, resembles that pigment quite as 
closely as the products obtained from bilirubin and hematin by the 
action of reducing agents; but no second band is developed when 
aldehyde is added to an alcoholic solution of urobilin. 1 

By the action of potassium permanganate upon urobilin Riva 
and Chiodera 2 obtained a substance closely resembling urochrome, 
and a similar product is formed when an aqueous solution of uro- 
bilin containing ether is evaporated upon a water bath. Neither 
product shows any absorption band, and both behave as urochrome 
does when it is acted upon by aldehyde. 

Uroerythrin. — Uroerythrin is the pigment which imparts the red 
color to crystals of uric acid and the pink tint to urate sediments. 
Under strictly normal conditions it probably does not occur in the 
urine, but it readily appears with the slightest deviation from health, 
and when present in larger amounts imparts a deep-orange color 
to the urine. Under pathological Conditions it is seen especially 
in cases of hepatic insufficiency, in which the liver, owing to a greatly 
increased destruction of red corpuscles, is unable to transform into 
bile pigment all the blood pigment which is carried to it. It also 
occurs when an absolute insufficiency on the part of the hepatic cells 
exists, so that the organ is not even capable of causing the transfor- 
mation of a normal amount of hemoglobin. Uroerythrin is seen in 
notable quantities in cases of cirrhosis and carcinoma of the liver, in 
passive congestion resulting from heart disease, in acute articular 
rheumatism, gout, pneumonia, malarial fever, erysipelas, spinal curva- 
ture, etc. In typhoid fever a marked excretion of uroerythrin is 
exceptional, and its occurrence has been associated with pulmonary 
complications. In nephritis it is seldom found in the urine, but 
Garrod cites an instance of pneumonia in which an abundant excre- 
tion of the substance accompanied conspicuous albuminuria. 

In certain diseases, such as hepatic cirrhosis, the excretion of 
uroerythrin, as also of urobilin, is said to be much diminished when 
the patient is placed upon a milk diet (Riva). 

When present in large amounts uroerythrin is readily recognized 
by the salmon-red color which it imparts to urinary sediments. 
Otherwise it is best to precipitate the urine with neutral lead acetate, 
barium chloride, or a similar reagent, when in the absence of uro- 
erythrin a milky- white precipitate is obtained, while a pale rose- 
colored sediment indicates the presence of the pigment in appreciable 
amounts; a more pronounced rose color is produced if large quan- 
tities are present. In every case at least ten to fifteen minutes should 

1 A. E. Garrod, "The Bradshaw Lecture on the Urinary Pigments in their 
Pathological Aspects," Lancet, Nov. 10, 1900. 

2 Arch. ital. di Clin. Med., 1896, vol. xxxv, p. 505. 



CHEMISTRY OF THE URINE 501 

be allowed to elapse before forming a definite conclusion, so that the 
sediment may have abundant time to settle. 

Normal Chromogens. — The chromogens occurring in normal urine 
are indican, urohematin, and an unknown chromogen which yields 
urorosein when treated with mineral acids. 

Indican. — It has been pointed out (see Sulphates) that the indol 
formed during intestinal putrefaction is oxidized to indoxyl in the 
blood; this, entering into combinaton with sulphuric acid, is elimi- 
nated in the urine as sodium or potassium indoxyl sulphate, or indican. 

Formerly it was thought that indican was also formed within 
the tissues of the body in the absence of putrefactive organisms (Sal- 
kowski) . 1 Further researches, however, have demonstrated that micro- 
organisms are always concerned in the production of indican, and that 
in health the large intestine is its sole source. Baumann, who 
succeeded in disinfecting the intestinal tract of a dog by means of 
large doses of calomel, observed that all traces of indican, as also of 
phenol and paracresol, disappeared from the urine. According to 
Senator, moreover, indican does not occur in the urine of newly born 
infants which have not as yet received nourishment. Tuczek's 
observations on abstinence from food in cases of insanity, in which 
indican was observed in the urine only when albumins, though in 
minimal amounts, were ingested, also speak very strongly against 
Salkowski's theory. Finally, it has been demonstrated that in cases 
in which an artificial anus is established near the distal end of the 
ileum the conjugate sulphates disappear almost entirely from the 
urine, while they reappear in normal amount as soon as the connection 
between the small and large intestines has been reestablished. 2 

The amount of indican which is normally eliminated in the urine 
varies somewhat with the character of the diet. Jarre 3 obtained 6.6 
mgrms. from 1000 c.c. of urine, as an average of eight observations. 
The largest quantities excreted in health are found after a liberal 
indulgence in animal food, while the smallest amounts are observed 
during a milk or kefir diet. By means of the latter article, indeed, 
the greatest diminution in the degree of intestinal putrefaction may 
be effected in man. 

In pathological conditions an increased elimination of indican is 
observed : 

1. In all diseases which are associated with an increased degree 
of intestinal putrefaction. As there appears to be little doubt that 
this is largely regulated by the acidity of the gastric juice, an in- 

1 Ber. d. deutsch. chem. Ges., 1876, vol. ix, pp. 138 and 408. Baumann, Zeit. 
f. physiol. Chem., 1886, vol. x, p. 123. Senator, Centralbl. f. d. med. Wiss., 
1877, vol. xv, pp. 357, 370, and 388. 

2 Nencki, Macfadyen u. Sieber, Arch. f. exper. Path u. Pharmakol., 1891, vol. 
xxix. 

3 Centralbl. f. d. med. Wiss., 1872, vol. x, pp. 2, 481, and 497; and Virchow's 
Archiv, 1877, vol. lxx, p. 72. 



502 THE URINE 

creased indicanuria, according to personal observations, is encountered 
when anachlorhydria or hypochlorhydria exists. Large quantities of 
indican are thus eliminated in cases of carcinoma of the stomach, and 
exceeded only by those observed in cases of ileus ; so that this symptom, 
in my estimation, is of considerable value in differential diagnosis, 
and is one, moreover, which has not received the attention it de- 
serves. Exceptions to this rule are at times, though rarely, met 
with, for which it is, however, impossible to account at present. 
Large quantities of indican are also observed in cases of acute, 
subacute, and chronic gastritis. In the course of personal obser- 
vations in this direction I was impressed with the curious phenome- 
non that in cases of ulcer of the stomach, notwithstanding the simul- 
taneous occurrence of hyperchlorhydria, an increased elimination of 
indican, contrary to what is usually seen in hyperchlorhydria refer- 
able to other causes, is quite commonly found. Possibly the exist- 
ence of muscular atony which was noted in these cases may serve to 
explain this apparent incongruity, but it is as yet impossible to offer 
a satisfactory explanation of the phenomenon. Remembering the 
origin of indican, and the relation which the amount eliminated bears 
to the degree of intestinal putrefaction, it will be unnecessary to 
enumerate the long list of diseases in which an increased indicanuria 
has been observed, as it will be found that in the majority of these 
cases the indicanuria is merely an index of the condition of the gastric 
juice and the motor power of the stomach. 1 

2. It should be noted that in cases in which the peristaltic move- 
ments of the small intestine have become impeded, as in ileus, acute 
and chronic peritonitis, an increased elimination of indican will inva- 
riably take place, no matter what the state of the gastric juice may 
be. In such conditions, and especially in ileus, the largest quanti- 
ties are observed, a point which may be of decided value in differ- 
ential diagnosis, as diseases of the large intestine alone are never 
associated with an increase in the amount of indican. In simple, 
uncomplicated constipation increased indicanuria is not seen; and 
should an examination in such cases reveal the presence of more 
indican than normal, it will be safe to assume the existence of disease 
elsewhere, and especially of the stomach. 

3. As albuminous putrefaction may also take place within the 
body, an increased indicanuria is observed in cases of empyema, 
putrid bronchitis, gangrene of the lung, etc.; but while in the con- 
ditions mentioned above the indol-producing organisms appear to be 
especially active, the elimination of phenol in the latter condition 
may be more pronounced at times than that of indican. Bearing in 
mind the points here set forth, I cannot agree with others in saying 

1 C. E. Simon, "Indicanuria," Am. Jour. Med. Sci. (full literature), 1895, vol 
ex, p. 48. 



CHEMISTR Y OF THE URINE 503 

that the study of indicanuria possesses no importance from a clini- 
cal standpoint. I maintain, on the other hand, that an examina- 
tion of the urine in this direction is at least as important as the testing 
for albumin and sugar, and that points of decided importance, not 
only in diagnosis, but also in treatment, may thus be gained. 

Of interest in this connection is the observation that in cases of 
increased indicanuria oxalate sediments are not uncommonly ob- 
served; but I am not willing to admit, as Harnack and van der 
Leyen suggest, that the indicanuria which follows the ingestion of 
small doses of oxalic acid is produced by a toxic action of the acid 
upon the tissue albumins. In these cases also the increased indican- 
uria is referable to increased intestinal putrefaction. 1 

When indican is treated with hydrochloric acid, it is decomposed 
into sulphuric acid and indoxyl; should an oxidizing substance be 
present at the same time, indigo blue, the blue coloring matter of 
the urine, results: 

2C 8 H 6 NKS0 4 + 20 = C 16 H ]0 N 2 O 2 + 2HKS0 4 . 
Potassium indoxyl Indigo blue, 

sulphate. 

Indigo blue in small amounts may be found free in the sediment of 
decomposing urines, usually occurring in the form of small, amorph- 
ous granules, more rarely in crystalline form. Urines have, however, 
also been observed which were blue when passed, or which turned 
blue as a whole upon standing. Such a phenomenon must be 
regarded as a medical curiosity. Undoubtedly it is referable to the 
action of microorganisms (see Bacteriuria), although McPhedran 
and Goldie mention that in their case bacteria were present only in 
small numbers. 2 

The blue pigment which may be obtained from urines has been 
variously described as Prussian blue, urocyanin, cyanurin, Harn- 
blau, uroglaucin, choleraic urocyanin, but it has been shown to be 
indigo blue, and derived from its colorless antecedent indican. This 
has been shown to be identical with the uroxanthin of Heller and 
Thudichum's choleraic urocyaninogen. 

Tests for Indican. — A few cubic centimeters of urine are mixed 
with an equal volume of Obermayer's reagent, and shaken with a 
small amount of chloroform, which last takes up the indigo blue which 
is formed. The resultant extract is normally either colorless or of a 
light sky blue; a darker color indicates an increased amount of 
indican. Obermayer 's reagent is a 2 pro mille solution of ferric chlo- 
ride in concentrated hydrochloric acid. 3 

1 v. Moraczewski, "Oxalurie and Indicanurie," Cent. f. inn. Med., 1903, No. 1. 

2 A. McPhedran and W. Goldie, "A Case of Indigosuria," Trans. Assoc. Am. 
Phys., 1901, vol. xvi, p. 242. 

3 Wien. klin. Woch., 1890, vol. iii, p. 176. 



504 THE URINE 

Stokvis' modification of Jaffe's test may also be employed. 1 To 
this end a few cubic centimeters of urine are treated with an equal 
volume of concentrated hydrochloric acid, and 2 or 3 drops of a 
strong solution of sodium or calcium hypochlorite. The mixture 
is shaken with 1 or 2 c.c. of chloroform as above. The indigo 
which is set free in this manner is taken up by the chloroform, and 
colors this blue to a greater or less extent, the degree of increase, as 
compared with the normal, being determined by the intensity of the 
color. Albumin need not be removed. Bile pigment, which inter- 
feres with the reaction, is removed by means of a solution of lead 
subacetate. Urines presenting a very dark color may be cleared in 
the same manner. Potassium iodide, owing to the liberation of free 
iodine, will color the chloroform a rose red. 

For the sake of comparison, it is well to employ the same quantities 
of urine and reagents in every case, marked tubes being very con- 
venient for this purpose. 

Quantitative Estimation. Wang's Method. 2 — The method is 
based upon the decomposition of potassium indoxyl sulphate by 
means of concentrated hydrochloric acid and the oxidation to indigo- 
blue of the indoxyl which is thus formed. The indigo blue is fur- 
ther transformed into indigo-sulphuric acid, and this titrated with 
a solution of potassium permanganate of known strength. 

Reagents required: 1. A 20 per cent, solution of lead acetate. 

2. Obermayer's reagent. This is a 2 pro mille solution of ferric 
chloride in concentrated hydrochloric acid (sp. gr. 1.19). 

3. Chloroform. 

4. Concentrated sulphuric acid. 

5. A mixture of equal parts of alcohol (96 per cent.), ether, and 
water. 

6. A solution of potassium permanganate containing about 3 grams 
pro liter The titration is conducted with this solution diluted in the 
proportion of 5 c.c. to 195 c.c. of water. Its titre is ascertained before 
each titration by comparing it with a dilute solution of oxalic acid of 
known strength; for example, one containing 0.1 gram of the acid dis- 
solved in 100 c.c. of water, as described on page 426. The amount 
of indigo blue which each cubic centimeter will represent is ascer- 
tained by multiplying the corresponding amount of oxalic acid by 1.04. 

Example. — Supposing that the permanganate solution is found of 
such strength that 1 c.c. represents 0.00014 gram of oxalic acid; 
the corresponding amount of indigo would be 0.00014 X 1.04= 
0.00015 gram. 

Method. — The urine is first examined for indican, as described 
above. Should a very intense reaction be thus obtained, only 25 or 

1 See Senator, Centralbl. f. d. med. Wiss., 1877, vol. xv, p. 257. 

2 "Ueber d. quantitative Bestimmung d. Harnindikans," Zeit. f. physiol. 
Chem., vol. xxv, p. 406. 






CHEMISTRY OF THE URINE 505 

50 c.c. are used for the quantitative estimation, while larger amounts 
are taken (200 to 500 c.c.) if the reaction is of only moderate intensity 
or negative altogether. 

The urine is precipitated with lead acetate solution, care being 
taken to avoid an excess. A large and accurately measured por- 
tion of the clear filtrate is treated in a separating funnel with an equal 
volume of Obermayer's reagent and extracted with chloroform. 
To this end 30 c.c. are added at a time and shaken for one minute. 
Two or three extractions are usually sufficient to remove the entire 
amount of indigo. The extract is placed in a small flask, and the 
chloroform distilled off. The residue is dried for a few minutes on 
a water bath until traces of remaining chloroform have been re-~ 
moved. It is then washed with the alcohol-ether-water mixture to 
remove the reddish-brown pigment which is present together with the 
indigo blue. The latter remains undissolved. After filtering off any 
particles of indigo that may be in suspension, through a small filter, 
this is dried and repeatedly extracted with boiling chloroform. The 
chloroform extract is filtered into the original indigo flask, the 
chloroform distilled off, the residue dried as before, and while still 
warm treated with 3 or 4 c.c. of concentrated sulphuric acid. The 
entire residue should be brought into solution by careful agitation. 
After standing or twenty-four hours the contents of the flask are 
poured into 100 c.c. of cold water; the flask is rinsed and the wash- 
ings added to the solution. This is filtered once more and titrated 
with the permanganate solution. At first the blue color of the solu- 
tion changes but little; later it turns greenish, and finally becomes 
yellowish or entirely colorless — not red. As a rule, the end reaction 
is quite distinct, but the titration requires experience. The best 
results are obtained if from 10 to 15 c.c. of the dilute permanga- 
nate solution are used. The resulting amount of indigo contained in 
the measured-off quantity of the first filtrate is then ascertained as 
described above. 

Example. — Amount of urine: 1780 c.c. 

The stock solution of potassium permanganate contains 3 grams 
to the liter; 1 c.c.=0.00596 gram of oxalic acid=0.0062 gram of indigo. 
Diluted solution (5 to 200); 1 c.c. =0.00015 gram of indigo. 300 c.c. 
of urine were precipitated with 25 c.c. of the lead solution; 250 c.c. 
of the filtrate, corresponding to 230.7 c.c. of urine, treated with 250 
c.c. of Obermayer's reagent. Extracted twice with chloroform. 4.3 
c.c. of the permanganate solution were used in the titration=0. 00065 
gram of indigo, correspondng to 0.005 gram in the 1780 c.c, accord- 
ing to the equation: 

230.7 : 0.00065 : : 1780 : x; x = 1A57 = 0.005, 

230.7 

Other methods for the quantitative estimation of indican which 
have heretofore been used, with the exception of the spectroscopic 



506 THE URINE 

method of Miiller, are not only inaccurate, but, like this, too time 
consuming and complicated to be of value to the practising physician. 
As a consequence almost all observers have based their conclusions 
upon an approximative estimation only. For practical purposes this 
is sufficient, and even Wang's method, though accurate and simple, 
will hardly find a ready entrance into the clinical laboratory, as it 
is still too time consuming and too expensive for daily use. 

Other quantitative methods are those of Ellinger 1 and Strauss, 2 
which should be read in the original. 

Urohematin. 3 — Urohematin appears to be the chromogen of the 
red pigment of the urine, and is very likely closely related to in- 
doxyl. Little is known of its chemical composition or of its mode 
of formation. In all probability the red pigment which may be 
obtained from this substance is identical with other red pigments 
which have been described from time to time as occurring in the 
urine, such as that of Scherer, the urrhodin of Heller, the urorubin 
of Plosz, Schunk's ind rubin, Bayer's indigo purpurin, Giacosa's 
pigment, and also the indigo red obtained by Rosenbach and Rosin 
by oxidation of the urine with nitric acid. 

Further investigations are necessary before this subject is fully 
understood; but bearing in mind the probable origin of urohematin 
from indoxyl, it would possibly be best to speak of the red pigment 
as indigo red. In accordance with the view that urohematin is an 
indoxyl derivative, its clinical significance is similar to that of indican 
(which see). 

Test. — The presence in normal urine of urohematin — i. e., a chromo- 
gen yielding a red pigment when treated with certain reagents — may 
be demonstrated by shaking urine with chloroform and decanting after 
several days, when the addition of a drop of hydrochloric acid to the 
chloroform extract will cause the appearance of a beautiful rose color; 
this varies in intensity according to the amount of the chromogen 
present. 

The purplish color so often obtained in the chloroform extract 
when Stokvis' modification of Jaffe's indican test is employed is due 
to a mixture of indigo blue and indigo red. Indican, however, is 
generally present in larger amounts than urohematin. In normal 
and, usually also, in pathological urines a red color is not obtained 
with the test mentioned. In a few isolated cases of ileus, peritonitis, 
and carcinoma of the stomach I have found more indigo red than 
indigo blue. 

The so-called " Reaction of Rosenbach" is a convenient test for 
indigo red when this is present in increased amounts; the boiling 
urine is treated drop by drop with concentrated nitr'c acid, when in 

1 Zeit. f. phys. Chem., vol. xxxviii, p. 178. 

2 Deutsch. med. Woch., 1902, April, 17. 

3 G. Harley, Verhandl. d. physik. med. Ges. z. Wiirzburg, 1855, vol. v, p. 1. 



CHEMISTRY OF THE URINE 507 

the presence of large amounts of indigo red it assumes a dark Bur- 
gundy color, which sometimes takes on a bluish tinge when held 
to the light. Owing to a precipitation of the pigment the mixture at 
the same time bee mes cloudy and the foam assumes a blue color. 
In well-marked cases the Burgundy colo does not appear to be 
changed by the further addition of nitric acid, but will sometimes 
suddenly change from red to yellow when 10 to 20 drops of the acid 
have been added. « 

Th'.s reaction Rosenbach 1 regarded as symptomatic of various 
forms of severe intestinal disease associated with an impeded resorp- 
tion throughout the entire intestinal tract. Ewald 2 likewise noted this 
reaction in cases of extensive disease of the small intestine, in carci- 
noma of the stomach, and in acute and chronic peritonitis; but he 
obtained negative results in carcinoma of the colon, stricture of the 
esophagus, chronic diarrhea, etc. Rosenbach's reaction should be 
viewed in the same light as a highly incresed elimination of indican. 
I have met with the reaction in all conditions associated with greatly 
increased intestinal putrefaction, and, like Ewald, failed to note the 
reaction in a few cases of occulsion of the large intestine, in which an 
increased elimination of indican is likewise never observed. 

Uroroseinogen. 3 — In addition to indican and urohematin, still 
another chromogen, which yields a rose-red pigment when treated 
with mineral acids, appears to occur in normal urine, although in 
small amounts. It is commonly regarded as a skatol derivative. 
The pigment, which has received the name urorosein, or Harnrosa, 
appears to be identical with Heller's urophain. Urorosein is best 
demonstrated by treating 5 to 10 c.c. of urine with an equal amount of 
concentrated hydrochloric acid, and 1 or 2 drops of a concentrated 
solution of sodium hypochlorite, when in the presence of much 
indican the mixture assumes a dark-greenish, blackish, or dark- 
blue color, owing to the formation of indigo. When the mixture 
is shaken with chloroform the supernatant fluid exhibits a beau- 
tiful rose color, which is due to the urorosein. This may now be 
extracted with amyl alcohol and separated from other pigments 
which are present at the same time, by shaking with sodium hydrate, 
whereby the solution is decolorized. Upon the addition of a drop 
or two of hydrochloric acid to the alcoholic extract the rose color 
reappears. Such solutions, however, soon become decolorized upon 
standing. A rose-red ring, referable to this pigment, is also fre- 
quently obtained in pathological urines when the ordinary nitric acid 
test is employed. 

While normally urorosein is obtained only in traces, appreciable 

1 Berlin, klin. Woch., 1889, vol. xxvi,.pp. 5, 490, and 520, and 1890, vol. xxvii, 
p. 585. 

2 Ibid., 1889, vol. xxvi, p. 953. 

3 H. Rosin, Deutsch. med. Woch., 1893, p. 51. 



508 THE URINE 

amounts are often met with in pathological conditions associated 
with grave disturbances oi nutrition, as in nephritis, diabetes, carci- 
noma, dilatation of the stomach, pernicious anemia, typhoid fever, 
phthisis, and at times in profound chlorosis, etc. A vegetable diet 
also appears to cause an increase in the amount of the chromogen. 

Pathological Pigments and Chromogens. The Blood Pigments. 
— The blood pigments proper which may occur in the urine have 
already been considered and in this connection it will only be necessary 
to refer briefly to the occasional presence of hematin, urorubro- 
hematin, and hematoporphyrin. 

Hematin is only rarely found. In order to demonstrate its pres- 
ence, the urine is rendered strongly alkaline with ammonia, filtered, 
and the filtrate examined spectroscopically. (See Blood.) 

Urorubrohematin and Urofuscohematin have been observed 
only once by Baumstark 1 in the urine of a case of pemphigus leprosus 
complicated with visceral lepra; they appear to be closely related to 
hematin. 

Hematoporphyrin. — McMunn found a pigment answering the des- 
cription of this substance in the urine in cases of rheumatism, Addi- 
son's disease, pericarditis, and paroxysmal hemoglobinuria, which 
he termed urohematin, but which in all probability was hematopor- 
phyrin. Le Nobel found the same pigment in two cases of hepatic 
cirrhosis and in one case of croupous pneumonia. Others have like- 
wise met with hematoporphyrinuria in various forms of hepatic dis- 
ease, as also in phthisis, exophthalmic goitre, typhoid fever, and 
hydroa aestivalis; further, in association with intestinal hemorrhages, 
in cases of lead poisoning, and especially during long-continued use 
of sulphonal, trional, and tetronal. Nebelthau records the history 
of a female patient, the subject of congenital syphilis, who had passed 
dark-red urine as long as she could remember, and continued to do so 
while under observation. Stern mentions a case in which marked 
hematoporphyrinuria was associated with icterus in a glucosuric 
individual. Recent researches, moreover, have shown that in traces 
at least the substance is present in every urine. As regards the origin 
of these normal .traces, the evidence is in favor of the view that they 
are formed within the body during its normal metabolism, and 
most likely in the liver, whence the substance is eliminated in the 
bile. A portion then escapes with the feces, while a similarly small 
amount is resorbed and eliminated in the urine. Increased amounts 
would accordingly suggest the existence of a hepatic insufficiency; 
and, as a matter of fact, we find that actual anatomical lesions then 
not infrequently occur. Taylor and Sailer thus report that in their 
case of sulphonal poisoning widespread degeneration of the hepatic 
cells existed; and Neubauer was able to isolate the pigment from the 

1 Pfliiger's Archiv, 1874, vol. ix, p. 568. See, also, J. W. Schultz, Diss., 
Greifswald, 1874. 



CHEMISTRY OF THE URINE 509 

liver of rabbits to which sulphonal had been administered, while it 
was absent in all other organs. On the other hand, it is difficult to 
ascribe all the phenomena of such hematoporphyrinuria to hepatic 
changes, seeing that changes of like degree may occur without con- 
spicuous urinary abnormality, and there is still much that is obscure 
in this condition. 

Stokvis attributed the increased elimination of hematoporphyrin 
in cases of lead poisoning and following the continued use of sul- 
phonal to the occurrence of hemorrhages into the intestinal mucosa, 
and suggested that the transformation of the hemoglobin into 
hematoporphyrin was favored by the sulphonal. But while intesti- 
nal hemorrhages may occur in the sulphonal cases, they are not 
always observed, and, as Garrod points out, Kast and Weiss, as 
also Neubauer, were unable to verify the recorded experiments of 
Stokvis, in which he claims to have obtained a small amount of 
hematoporphyrin when fresh blood was digested with pepsin-hydro- 
chloric acid and sulphonal at from 38° to 40° C. 

Urines which contain much hematoporphyrin are usually dark 
red in color, but the shade may vary from a sherry or port-wine 
tint to a dark Bordeaux. It is noteworthy, however, that this color 
is not primarily due to the exaggerated degree of hematoporphy- 
rinuria, but, as Hammarsten first pointed out, to other abnormal 
pigments which are but little known, but which are probably closely 
related to hematoporphyrin. As Garrod says, the removal of 
the hematoporphyrin from such urines causes little or no change 
of color, and when this pigment is added to normal urine until on 
spectroscopic examination bands of similar intensity are seen, the 
change of tint produced is comparatively slight. In one such case, 
not due to sulphonal, he was able to isolate a purple pigment which 
differed in its properties from any known urinary coloring matter, 
and to which the color of the urine in question was obviously in the 
main due. Neumeister also states that in sulphonal intoxication an 
iron-containing derivative of hemoglobin occurs in the urine, which 
presents a reddish-violet color and shows a single band of absorption 
in the blue portion of the spectrum immediately bordering on the green. 

Albumin is not present in uncomplicated cases of hematopor- 
phyrinuria, and the pigment itself does not give the albumin reactions. 

To demonstrate the presence of hematoporphyrin under normal 
conditions, or when small amounts only are present in the urine, 
Garrod's method should be employed. 

Garrod' s Method. — Several hundred c.c. of urine (500 to 1500) are 
treated with a 10 per cent, solution of sodium hydrate in the propor- 
tion of 20 c.c. of the alkali solution for 100 c.c. of urine. The pre- 
cipitated phosphates are filtered off and thoroughly washed by 
repeatedly suspending them in water. Should the precipitate be of 
a reddish color, or if it shows the spectrum of hematoporphyrin in 



510 THE URINE 

alkaline solution when examined on the filter in the moist state, we 
may conclude that much hematoporphyrin is present. In this case 
it is washed until the filtrate is colorless. If traces only are 
present, however, one washing must suffice. The precipitate is 
then treated with alcohol, which is acidified with hydrochloric acid 
to such an extent that the phospates are entirely dissolved. The 
resulting solution should not exceed 15 to 20 c.c. in volume. This 
is ihen examined in a layer, of not less than 3 to 4 cm. in thickness, 
for the spectrum of acid hematoporphyrin, using a spectroscope 
with slight dispersion. The solution is now rendered alkaline with 
ammonia and treated with an amount of acetic acid which just suffices 
to redissolve the precipitated phosphates. On shaking with chloro- 
form this extracts the pigment, and the chloroform solution then gives 
the spectrum of the alkaline hematoporphyrin, since organic acids 
do not change the pigment to the form which yields the acid spectrum. 
The residue which remains after evaporating the chloroform can 
finally be washed with water and dissolved in alcohol, when a nearly 
pure solution is obtained, which is comparable with a solution of 
hematoporphyrin obtained from hematin. 

Precautions: If a preliminary test shows that the urine con- 
tains but little phosphates, a small quantity of calcium phosphate 
in acetic acid is added before the urine is rendered alkaline with the 
sodium hydrate solution. As hematin and chrysophanic acid are 
also precipitated with the phosphates, their absence must be ensured. 
For this reason the urine should contain no rhubarb or senna. 

In conclusion, it may be said that a chromogen of hematopor- 
phyrin is also usually present in urines containing the free pigment, 
which probably explains why such urines gradually become darker 
on standing. 

Literature. — A complete account of the literature on hematoporphyrinuria 
up to 1893 is given by R. Zoja, "Su gualche pigmento di alcune urine," etc., 
Arch. ital. di clin. med., 1893, vol. xxxii, p. 63. A. E. Garrod, loc. cit.; and 
Centralbl. f. inn. Med., 1897, No. 21. Taylor and Sailer, Contributions from the 
William Pepper Laboratory, Philadelphia, 1900, p. 120. O. Neubauer, Arch. f. 
exper. Path. u. Pharmakol., 1900, vol. xliii, p. 455. B. J. Stokvis, " Zur Patho- 
genese d. Hsematoporphyrinurie," Zeit, f. klin. Med., vol. xxviii, p. 1. Kast u. 
Weiss, Berlin, klin. Woch., 1896, vol. xxxiii, p. 621. Hammarsten, "Skandin. 
Arch. f. Physiol.," 1891, vol. hi, p. 31. Neumeister, Physiol. Chem., Jena, 1897. 
Nebelthau, Zeit. f. physiol. Chem., 1899, vol. xxvii, p. 324. B. Ogden, Boston 
Med. and Surg. Jour., 1898 

Biliary Pigments. — Of the four biliary pigments, viz., bilirubin, 
biliverdin, biliprasin, and bilifuscin, the former alone is met with in 
freshly voided urines, while the others may form upon standing, 
being oxidation products of bilirubin. The pigment is never found 
in normal urine, and its occurrence may be regarded as a positive 
symptom of disease. 

In health it will be remembered that bilirubin is formed in the 
liver from blood pigment, and is eliminated into the small intestine, 



CHEMISTRY OF THE URINE 511 

in which it is transformed into hydrobilirubin and largely excreted as 
such in the feces, while a small portion is reabsorbed into the blood 
and eliminated in the urine as urochrome or normal urobilin. When- 
ever, then, the outflow of bile into the intestines becomes impeded 
bilirubin is absorbed by the lymphatics and eliminated in the urine. 

Among the numerous causes which give rise to choluria under 
such conditions may be mentioned obstruction of the biliary ducts, 
and especially of the common duct, referable to simple swelling of 
its mucous membrane, as in the ordinary forms of catarrhal jaun- 
dice. It may also be due to the presence of a biliary calculus, to 
parasites, compression of the duct by tumors of the liver, the gall 
bladder, the duct itself, and of neighboring structures, and particu- 
larly of the pancreas, stomach, and omentum. Whenever the 
blood pressure in the liver is lowered, so that the tension in the 
smaller biliary ducts becomes greater than that in the veins, choluria 
likewi e results. The icterus occurring under all such conditions 
has been termed hepatogen c icterus, in contradistinction to the form 
observed in cases in which the liver has either totally or partially 
lost the power of forming bile, be this owing to the existence of 
degenerative processes affecting its glandular epithelium, as in cases 
of acute yellow atrophy, or to destruction of red corpuscles 
going on so rapidly and so extensively that the organ is incapable of 
transforming into bilirubin all the blood pigment which is carried to 
it. This occurs in some cases of pernicious anemia, malarial intoxi- 
cation, typhoid fever, poisoning with arsenious hydride, etc. Icterus 
neonatorum is probably to a certain extent also dependent upon the 
latter cause. To this form the term hematogenic icterus has been 
applied. In such cases the occurrence of bilirubin in the urine can 
only be explained by assuming that a transformation of blood-color- 
ing matter into bilirubin has taken place in the blood itself or in other 
tissues of the body. As a matter of fact, it appears to be generally 
accepted that such a trans ormation can occur outside of the liver, as 
the hematoidin which may be found in old extravasations of blood 
seems to be identical with bilirubin. On the other hand, however, 
the existence of a hematogenic icterus is positively denied, especially 
by Stadelmann. In accordance with his view it may be demon- 
strated that in cases of pernicious anemia, malaria, etc., the urine 
does not contain bilirubin, but usually urobilin. In cases of this 
kind which I had occasion to examine, bilirubin was, as a matter of 
fact, never found. Further investigations are necessary to settle this 
question. 

Usually the presence of biliary pigment may be recognized by 
direct inspection, as urines which contain it in notable amounts 
present a color varying from a bright yellow to a greenish brown. 
Any morphological elements which may occur in the sediment are 
stained a golden yellow, and the same color is imparted to the foam 



512 THE URINE 

of the urine as well as to the filter paper used in the filtration. At 
times, however, and particularly in cases in which the icterus is only 
beginning to appear, the presence of bilirubin is not infrequently 
overlooked, and urines containing urobilin in large amounts may be 
similarly mistaken for icteric urines. In doubtful cases, therefore, 
whether icterus exists or not, but in which the urine presents an 
intense yellow color, it is necessary to have recourse to chemical 
tests. A large number of these have been devised, all of which are 
fairly reliable. Only those will be described which I have examined 
myself and which are especially delicate. 

Smith's Test 1 — 5 to 10 c.c. of urine are placed in a test-tube 
and treated with 2 or 3 c.c. of tincture of iodine (which has been 
diluted with alcohol in the proportion of 1 to 10) in such a manner 
that the iodine solution forms a layer above the urine. In the pres- 
ence of bilirubin a distinct emerald-green ring is seen at the zone of 
contact. This test can be highly recommended, as it is exceedingly 
simple and not surpassed in delicacy by any other. 

Huppert's Test. 2 — 10 to 20 c.c. of urine are precipitated with 
milk of lime (a solution of barium chloride is, perhaps, still more 
convenient), and the precipitate after filtering brought into a beaker 
by perforating the filter and washing its contents into the latter with 
a small amount of alcohol acidualted with sulphuric acid. The 
mixture is boiled, when in the presence of bilirubin the solution 
assumes a bright emerald-green color. Huppert's test is as delicate 
as is that of Smith, but is not so convenient for the needs of the 
practising physician. 

Gmelin's Test (as modified by Rosenback). 3 — The urine is filtered 
through thick Swedish filter paper, when the latter is removed and 
a drop of concentrated nitric acid, which has been allowed to stand 
exposed to the air for a short time, is placed upon its inner surface. 
In the presence of bilirubin a prismatic play of colors will be seen 
to occur around the nitric acid spot. 

Gmelin's Test.* — The urine is treated with nitric acid, which is 
carried to the bottom of the test-tube by means of a pipette, so as 
to form a layer beneath the urine, when a color play, as already 
described (p. 463), will take place at the line of contact between 
the two fluids; the green color is the most characteristic. 
fc' In this connection a few words may also be said of the occurrence 
in^the urine of biliary acids and cholesterin. 

Biliary Acids. — These jnay usually be found in the urine whenever 
bile pigment is present, so that their clinical significance is essen- 

B l Dublin Med. Jour., 1876, p. 449. 

I 2 Arch. d. Heilk., 1867, vol. viii, pp. 351 and 476. 

I 3 Centralbl: f. d. med. Wiss., 1876, vol. xiv, p. 5. 

^Tiedemann u. Gmelin, Die Verdauung nach Versuchen, Heidelberg, 1826, 
^IP-ISO- 



CHEMISTRY OF THE URINE 513 

tially the same as that attaching to bilirubin. Their demonstration is, 
however, attended with much difficulty (see Feces). 

Cholesterin. — Cholesterin has never been found in icteric urines, 
and is only rarely seen in other pathological conditions. It has 
been observed in cases of chyluria, fatty degeneration of the kidneys, 
diabetes, in one case of epilepsy, in eclampsia, and in several cases 
of pregnancy, v. Jaksch noted cholesterin crystals in a urinary sedi- 
ment in a case of tabes and cystitis. Glinsky records a similarobser- 
vation. Harley found it repeatedly in cases of pyuria. Reich states 
that he found cholesterin crystals of the size of a dollar in the urine of 
a case of chronic cystitis. Hirschlaff found larger quantities in the 
urine of a case of hydronephrosis; on one occasion 5.8 grams in 100 
c.c. of urine. I have found cholesterin crystals in the sediment in 
a case of acute nephritis. Giiterbock described a urinary calculus 
obtained from the bladder of a woman which consisted almost 
entirely of cholesterin (see also Feces \ Langgaard noted the pres- 
ence of the substance in a case of chyluria. 1 

Pathological Urobilin. — This pigment should not be confounded 
with the urochrome or normal urobilin described above, to which 
it is closely related, but from which it may be distinguished by means 
of the spectroscope. Gautier states that pathological urobilin may 
be obtained from urochrome by submitting the latter to the action 
of reducing agents; and, as I have already pointed out, Riva and 
Chiodera obtained a substance from urobilin by the action of potas- 
sium permanganate, which closely resembles urochrome. It is said 
to be identical with the stercobilin found in the feces, but differs 
from Maly's hydrobilirubin in containing a much smaller percentage 
of nitrogen, viz., 4.11, as compared with 9.22 (Garrod and Hop- 
kins). While its occurrence in the urine is essentially a pathological 
phenomenon, it is at times also met with in normal urine, and 
appears to be derived from a special chromogen, urobilinogen, from 
which it may be set free by the addition of an acid. Both urobilin 
and its chromogen are precipitated by saturating the urine with 
ammonium sulphate, and both are soluble in chloroform. Accord- 
ing to Maly, urobilin is formed by the reduction of bilirubin in the 
intestine, and is then in part resorbed and eliminated in the urine. 
Hayem, on the other hand, proposed the hypothesis that the sub- 
stance originates in a diseased or disordered liver, as bilirubin does 
in the same organ in health, and accordingly he regards the appear- 
ance of much urobilin in the urine as evidence of hepatic insuf- 
ficiency. Others, again, maintain that urobilin is formed in the 
tissues at large either by the reduction of bilirubin or directly from 
the blood pigment. The first view is notably held by Kunkel, Mya, 

1 v. Jaksch, Klinische Diagnostik, 4th ed. ; p. 339. Glinsky, Maly's Jahresber., 
1894, vol. xxiii, p. 484. Langgaard, Virchow's Archiv, vol. lxxxvi. W. Hirs- 
chlaff, Deutsch. Arch., 1899, vol. lxii, p. 53. 
33 



514 THE URINE 

Giarre, and others, while the hematogenous theory is notably 
represented by Gerhardt. Garrod discusses these various hypotheses 
at some length in his most interesting lecture on the urinary pig- 
ments in their pathological aspects, in which he personally inclines 
to the intestinal theory, as now held by Muller, Schmidt, Esser, and 
others. In a work of this scope it would lead too far to discuss 
the various investigations which lend themselves in support of this 
view, and I can here quote only the following from Garrod's paper: 
"The chief seat of the formation of urobilin (for it is convenient to 
employ this term as including both pigment and chromogen) is 
undoubtedly the intestinal canal. This can only be gainsaid by 
denying the identity of the urinary and fecal pigments. The quan- 
tity normally present in the feces is far larger than that which enters 
the intestine with the bile (when a small amount is found), and 
there is strong evidence that the urobilin in bile is itself of intesti- 
nal origin. This being so, it is clear that theories other than the 
intestinal and its modifications merely attempt to trace a second 
source for the urobilin of the urine. It is equally clear that the 
substance from which the intestinal urobilin is formed is the bile 
pigment. Under ordinary conditions the bile pigment is destroyed 
in its passage along the intestine, and does not appear as such in 
the feces. In its place we find large quantities of urobilin, which 
in its turn disappears when occlusion of the common duct prevents 
the entrance of bile into the intestine. Again, when under certain 
morbid conditions the bile pigment passes along the intestine unal- 
tered, urobilin is absent from the feces. However, the conversion 
of bilirubin into urobilin is no mere process of reduction, but in- 
volves a much more radical change, with elimination of nitrogen. 
That the change is brought about by bacterial action there is much 
evidence to show. When bile is inoculated with fecal material and 
kept in an incubator a formation of urobilin rapidly takes place, and 
at the same time the bile pigment diminishes, and ultimately dis- 
appears." 

From its frequent occurrence in febrile urines pathological urobilin 
has also received the name febrile urobilin. 

Its presence is very common in hepatic cirrhosis. In 12 cases of 
the atrophic and hypertrophic variety v. Jaksch was able to demon- 
strate urobilin in every instance, a point which may at times be of 
considerable diagnostic importance. I have observed urobilin in a few 
cases of hepatic cirrhosis, chronic malaria, and pernicious anemia, in 
all of which the skin presented a light icteric hue, and in which bile 
pigment was absent from the urine. Unfortunately, an examination 
of the blood was not made, and I have hence not been able to con- 
firm the statement of v. Jaksch that bilirubin occurs in the blood 
in almost every case in which urobilin is present in the urine. Syl- 
laba, however, has shown that in pernicious anemia, urobilinuria 






CHEMISTRY OF THE URINE 515 

is quite constantly associated with bilirubinemia (see the latter). 
Urobilin has also been noted in cases of carcinoma, scurvy, Addi- 
son's disease, hemophilia, in cases of retro-uterine hematocele, in 
extra-uterine pregnancy, following intracranial hemorrhages, etc. 
According to Bargellini, the degree of constipation in simple atony 
of the bowel is without influence upon the amount of urinary uro- 
bilin, but he states that in typhoid fever it causes an obvious increase; 
whereas disinfection or emptying of the large bowel produces a notable 
diminution in the amount. Urobilinuria, according to Samberger, 1 
is common early in secondary syphilis and referable to increased 
destruction of red cells. In some cases the urobilinuria is only 
observed after the mercurial treatment has been instituted, and sub- 
sequently disappears. 

Urines rich in urobilin usually present a dark-yellow color which 
is strongly suggestive of the presence of bilirubin; even the foam 
in such cases may be colored, making the resemblance between the 
two pigments still more complete. This dark color, however, is not 
due to urobilin, but to associated pigments. 

Gerhardt's Test. — If the urine contains much urobilin, which 
the color will indicate, 10 to 20 c.c. are extracted with chloroform by 
shaking, and the extract treated with a few drops of a dilute solu- 
tion of iodopotassic iodide. Upon the further addition of a dilute 
solution of sodium hydrate the chloroform extract is colored a yellow 
or yellowish brown, and exhibits a beautiful green fluorescence; this 
is even more intense than that noted in the case of normal urobilin. 

Bratjnstein's Test. — The reagent is composed of 100 c.c. of a con- 
centrated solution of copper sulphate, 6 c.c. of concentrated hydro- 
chloric acid, and 3 grams of ferric chloride; 20 c.c. of urine are 
treated with 3 to 4 c.c. of the reagent and shaken with chloroform. 
In the presence of urobilin a rose to a red color develops. 

Schlesinger's Test. — 10 c.c. of urine are treated with an equal 
quantity of a 1 per cent, solution of acetate of zinc in absolute alcohol. 
The mixture is agitated and filtered, when in the presence of urobilin 
the filtrate will show distinct fluorescence. 

Spectroscopic Examination. — The urine is best examined as 
follows: 50 c.c. of urine are extracted in a separating funnel with 
amyl alcohol, which takes up both the pigment and its chromogen. 
After standing for several hours the urine is allowed to flow away 
by opening the stopcock, when the alcoholic extract is decanted from 
above, and is treated with a concentrated alcoholic and ammoniacal 
solution of zinc chloride. In the presence of urobilin the liquid 
shows a beautiful fluorescence, and on spectroscopic examination a 
single band of absorption is seen between b and F. In acid solu- 
tions, on the other hand, a single band is likewise obtained between b 

1 Arch. f. Dermat. und Syph., 1903, vol, lxvii. 



516 THE URINE 

and F, but this extends to the right beyond F, and is much darker. 
Should the urine contain much urobilin, its special extraction is not 
necessary. In such an event the acid urine shows the acid spectrum, 
while the alkaline band is obtained after the addition of ammonia. 
(See also Bang's Test.) 

Literature. — A. E. Garrod, loc. cit. A. E. Garrod and F. G. Hopkins, " On 
Urobilin," Jour, of Physiol., 1898, vol. xxii, p. 451. Maly, Centralbl. f. d. med. 
Wiss., 1871, vol. ix, p. 849. Hayem, Gaz. hebdom., 1887, vol. xxiv, pp. 520 and 
534; and Gaz. des hop., 1889, vol. lxii, p. 1314. Kunkel, Virchow's Archiv, 
1880, vol. lxxix, p. 655. Mya, Arch. ital. di clin. med., 1891, vol. xxx, p. 101; 
and Lo Sperimentale, 1896, vol. 1, p. 71. Giarre, ibid., 1895, vol. xlix, p. 89,. and 
1896, vol. 1, p. 81. F. Miiller, Schlesische Gesellsch. f. vaterland. Kultur, Janu- 
ary, 1892. A. Schmidt, Verhandl. d. XIII Congress, f. inn. Med., 1895, p. 320. 
Esser, Untersuchungen iiber d. Entstehungsweise d. Hydrobilirubins, etc., Diss. 
Bonn., 1896. Bargellini, Lo Sperimentale, 1892, vol. xlvi, p. 119. v. Jaksch, 
Zeit. f. Heilk., 1895, vol. xvi, p. 48. D. Gerhardt, Zeit. f. klin. Med., 1897, vol. 
xxxii, p. 313. 

Melanin and Melanogen. — In cases of melanotic disease it has been 
repeatedly observed that the urine, which usually and probably 
always presents a normal yellow color when voided, gradually be- 
comes darker upon exposure to the air, and finally turns black. 
Such urines generally contain melanin and its chromogen in solution; 
deposits of melanin granules by themselves are only occasionally 
seen, and are not characteristic, as they may also be found in cases of 
chronic malarial intoxication, etc. 

While the occurrence of melanin in the urine is probaly indica- 
tive in most cases of the existence of melanotic tumors, it should 
be stated that this symptom cannot be regarded as pathognomonic, 
as it may be absent in the case of melanotic tumors, and present in 
wasting diseases and inflammatory affections, and may at times, 
though very rarely, be associated with non-pigmented growths. 
Nevertheless, its occurrence should always be regarded with suspicion, 
and, taken in conjunction with other symptoms, will often lead to a 
correct diagnosis. 

Tests for Melanin and Melanogen. — 1. The presence of 
melanogen may be assumed if upon the addition of ferric chloride 
solution a black precipitate appears in the urine, which is soluble in 
a solution of sodium carbonate, and can be reprecipitated as a black 
or brownish-black powder by mineral acids. Instead of ferric chlo- 
ride barium hydrate may also be used. 

2. A few cubic centimeters of urine are treated with bromine- 
water, when in the presence of melanin or melanogen a precipitate is 
obtained, which is yellow at first, but gradually turns black. 

Literature. — T. H. Eiselt, "Die Diagnose d. Pigmentkrebses durch d. Harn," 
Prag. Vierteljahrschr. f. praktische Heilk., 1858, iii, p. 190, and 1862, vol. iv, 
p. 26. Senator, "Ueber schwarzen Urin," Charite Annal., 1891. Hoppe-Seyler, 
Zeit. f. physiol. Chem., 1891, vol. xv, p. 179. F. Grohe, " Zur Gesch. d. Mela- 
naemie," Virchow's Archiv, 1861, vol. xx, p. 306. 



CHEMISTRY OF THE URINE 517 

Phenol. — Phenol, according to Brieger, occurs only in very small 
amounts in human urine, the usual phenol reactions being largely 
referable to paracresol. Normally, about 0.03 gram is eliminated 
in the twenty-four hours, but in pathological conditions much larger 
quantities may be found. Remembering the origin of phenol, it is 
clear that an increased elimination may be observed whenever putre- 
factive processes are going on in the tissues and cavities of the body, 
or whenever there is an increase in the degree of intestinal putre- 
faction, though in the latter condition the indican is usually the only 
conjugate sulphate that is found increased. In peritonitis, diph- 
theria, erysipelas, scarlatina, empyema, pulmonary gangrene, putrid 
bronchitis, etc., an increased elimination of phenol is commonly 
seen, as also in certain cases of pernicious vomiting of pregnancy. 
Important from a diagnostic standpoint, further, is the fact that in 
uncomplicated cases of typhoid fever no increase is observed, while 
this is common in tuberculous meningitis. 1 The largest amounts, 
of course, are seen in cases of poisoning with carbolic acid or one of 
its derivatives (hydroquinone, pyrocatechin, salicylic acid), where 
the urine may darken on standing, thus simulating true melanuria. 

As the quantitative estimation of phenol is too complicated for the 
purposes of the general practitioner, Salkowski's qualitative test is 
here also described. From the intensity of the reaction certain con- 
clusions may be drawn as to the amount present. It is especially 
serviceable in cases of suspected poisoning with carbolic acid. 

Salkowski's Test. — About 10 c.c. of urine are boiled in a test- 
tube with a few cubic centimeters of nitric acid, and, on cooling, 
treated with bromine-water. The development of a pronounced 
turbidity or the occurrence of a precipitate indicates the presence 
of an increased amount of phenol. 

Quantitative Estimation. Principle. — When potassium-phenyl 
sulphate is treated with hydrochloric acid, phenyl sulphate results, 
which further takes up one molecule of water, giving rise to the 
formation of sulphuric acid and phenol. 

From the action of bromine-water upon phenol a yellowish-white 
crystalline precipitae of tribromophenol results: 

C 6 H 5 .OH + 6Br = 3HBr + C 6 H 2 Br3.0H. 

As 331 (molecular weight) parts by weight of tribromophenol 
correspond to 94 (molecular weight) parts by weight of phenol, the 
amount of the latter contained in a certain volume of urine is readily 
determined according to the equation 

331: 94:: x: y; and y = ^k x = 0.28398 x, 
v, u 331 

1 A. Strasser, "Ueber d. Phenolausscheidung bei Krankheiten," Zeit. f. klin. 
Med., vol. xxiv, p. 543. Brieger, Zeit. f. klin. Med., 1881, vol. iii, p. 468. Kast 
u. Baas, Munch, med. Woch., 1888, vol. xxxv, p. 55. 



518 THE URINE 

in which x indicates the weight of the tribromophenol found in the 
amount of urine employed, and y the corresponding quantity of 
phenol. 

Method. — From 500 to 1000 c.c. of urine are treated with one-fifth 
of an equivalent amount of dilute hydrochloric acid (1 to 4), and dis- 
tilled so long as a specimen of the distillate is rendered cloudy upon the 
addition of bromine-water (1 to 30), the specimens used for this pur- 
pose being carefully preserved. The total quantity of the filtered dis- 
tillate, together with the specimens which have been set aside, is now 
treated with bromine-water, shaking the mixture after each addition 
of the reagent until a permanent yellow color results. Beyond this 
point further addition is beset with danger, as compounds will be 
formed which contain more bromine, the presence of which would 
indicate a smaller amount of phenol than that actually contained in 
the urine. After two or three days the precipitate is collected on a 
filter which has been dried over sulphuric acid, washed with water 
containing a trace of bromine, and then dried over sulphuric acid 
and weighed. 

Salol and salicylic acid may be recognized from the fact that such 
urines when treated with a solution of ferric chloride develop a 
marked violet color which does not disappear on standing. The 
reaction thus differs from that obtained with diacetic acid. 

Alkapton. — Urines are at times, though very rarely, seen which, 
like the phenol urines, turn dark on standing, but in which the 
change in color is neither referable to the presence of phenol or its 
derivatives, nor to melanin. Such urines are of a normal color when 
passed, but gradually turn reddish brown upon exposure to the 
air. Treated with a small amount of alkai, this change occurs 
almost immediately. Fehling's solution is reduced on the applica- 
tion of heat, while bismuth is not affected. Ammoniacal silver 
solution is reduced in the cold, and a temporary bluish-green color 
develops when the urine is treated with a ferric salt. The fermenta- 
tion test is negative, and examination with the polarimeter shows 
that the substance in question is not glucose. With phenylhydrazin 
no osazone is formed. 

Bodeker, who first observed a urine of this kind, termed the sub- 
stance giving rise to the reactions just described alkapton, and sub- 
sequently expressed the belief that his alkapton might possibly have 
been pyrocatechin. Subsequent investigators succeeded in isolating 
substances from such urines which have been variously termed pyro- 
catechuic acid, urrhodinic acid, glucosuric acid, uroleucinic acid, and 
uroxanthinic acid. Baumann and Wolkow later were able to iso- 
late homogentisinic acid in pure form from the urine of such cases, 
and expressed the belief that some of the substances obtained by 
previous observers were in reality the same. Since that time this 
acid has also been found by Garrod, Ogden, Stange, Stier, and others. 






CHEMISTRY OF THE URINE 519 

Of the origin of alkapton little is known. Baumann expressed 
the opinion that homogentisinic acid might be derived from tyrosin, 
and that the condition is referable to the activity of special micro- 
organisms in the upper portions of the intestines. As a matter of 
fact the amount of homogentisinic acid can be materially increased 
by the administration of tyrosin, and Mittelbach has shown that 
if the substance is given in frequently repeated and small doses 
almost the entire amount reappears in the urine as homogentisinic 
acid. Tyrosin, however, belongs to the para-series, while homogen- 
tisinic acid is an or^o-compound, so that the transformation of tyro- 
sin into homogentisinic acid cannot be a direct process, and it has 
accordingly been questioned whether Baumann's view regarding the 
origin of alkapton is correct. There is evidence indeed to show that 
homogentisinic acid does not originate in the intestines, viz., is not a 
product of bacterial activity. It has thus been found that the alkap- 
tonuria does not cease during starvation, and that a restriction of the 
putrefactive processes in the intestines by means of oil of turpentine, 
a kefir diet, and the administration of /3-naphthol does not lead 
to a diminished elimination of homogentisinic acid. It has never 
been found in the feces, moreover, and Garrod has shown that after 
inoculation of common bouillon, meat juice, or tyrosin broth with 
alkaptonuric feces homogentisinic acid is not formed. On the other 
hand, Embden observed that when an alkaptonuric individual took 
homogentisinic acid by the mouth a far larger portion appeared in 
the urine than when the same substance was administered to a healthy 
individual, which suggests that the alkaptonuria may be referable to 
impairment of the normal processes of oxidation. Very significant is 
the discovery that a notable increase follows the administration of 
phenylalanin, and that the ingestion of phenylacetic acid will increase 
the power of reduction and of rotation of the urine. Phenylpro- 
pionic acid and benzoic acid cause no increase in the elimination of 
homogentisinic acid. 

The prevailing view is that alkaptonuria is a metabolic anomaly 
comparable to glucosuria and cystinuria; but, unlike glucosuria, it 
can scarcely be regarded as an expression of a pathological process. 
It may, of course, occur in individuals, suffering from disease, and 
has been observed in connection with glucosuria, in acute gastro- 
intestinal catarrh, in phthisis, acute miliary tuberculosis, in one case 
of brain tumor, carcinoma of the prostate, etc. More frequently the 
condition is accidentally discovered in apparently healthy individuals, 
and has repeatedly been confounded with glucosuria owing to the 
positive reduction test with Fehling's solution. 

Garrod, from an analysis of all the reported cases, concludes that 
the condition is nearly always congenital. In 32 known instances 
which were presumably congenital, 19 occurred in seven families. 
One family contained 4 alkaptonurics, three others 3, and the re- 



520 THE URINE 

maining three 2 each. In fully 60 per cent, of the cases, it appears 
from Garrod's studies, the parents of alkaptonurics were first cousins. 
There is thus far only one known instance in which ihe anomaly has 
been transmitted by an alkaptonuric father to his son. 

The condition commonly persists through years and perhaps a life- 
time. It may also occur as a transitory abnormality, however, as is 
apparent from the case of Hirsch, in which the condition persisted 
for three days, and the case of Geyger, in which the alkaptonuria was 
observed on only two days. A few observers further report the 
occurrence of alkaptonuria shortly preceding death. 

Very interesting in this connection is the observation of Osier and 
others that the urine of patients with ochronosis will darken on stand- 
ing and may contain homogentisinic acid. The pigmentation of the 
cartilages thus seemed to be a possible morphological expression of the 
urinary abnormality. But as Garrod has already stated, it is possible 
also that other substances besides homogentisinic acid may cause the 
blackening of the urine in ochronosis. 

The amount of homogentisinic acid eliminated in the twenty-four 
hours is variable, but usually large. Baumann found an average 
elimination of 4.6 grams; the largest amount in twenty-four hours 
was 6 grams. In Meyer's case, a child one and one-half years old, 
3.3 grams were passed pro die. Larger quantities are obtained after 
a liberal diet of meats than with a vegetable diet. 

Isolation and Estimation (Garrod's Method). — The urine is 
heated nearly to boiling without any preliminary treatment, and for 
each 100 c.c. at least 5 or 6 grams of solid neutral lead acetate are 
added. 

As soon as the acetate is dissolved, the bulky gray precipitate 
which forms is removed by filtration, and the filtrate, which has a 
pale-yellow color, is allowed to stand for twenty-four hours in a cool 
place. If the urine be very rich in homogentisinic acid, or if the 
flask containing the filtrate be placed upon ice, minute acicular 
crystals, which are almost colorless, quickly form; but as a rule 
crystallization does not commence until several hours have elapsed. 
The crystals are then much larger, are grouped in stars or rosettes, 
and are more deeply colored. 

In summer weather it would probably be desirable to start the 
crystallization by artificial cooling; but although the process is greatly 
accelerated at a low temperature, the total yield is not materially 
increased. 

If the formation of the crystals be long delayed, the liquid may be 
warmed again and more lead acetate added. 

After the lapse of twenty-four hours crystals cease to form, even 
when the liquid is placed upon ice. 

The crystalline product so obtained is lead homogentisinate. When 
the crystals are dissolved in hot water the solution assumes a deep- 



CHEMISTRY OF THE URINE 521 

brown color with alkalies; it reduces Fehling's solution readily with 
the aid of heat, and yields a transitory deep-blue color with a dilute 
solution of ferric chloride. From the lead salt free homogentisinic 
acid may be obtained by decomposing it with hydrogen sulphide. 
For clinical purposes the following method also may be employed: 
Baumann's Method. — 50 c.c. of urine are treated with 15 grams of 
ammonium chloride, which should be brought into solution by shaking, 
in a stoppered graduate. After standing for about twelve h©urs to 
allow the uric acid to separate out the solution is filtered and an 
accurately measured portion of the filtrate titrated with a decinor- 
mal ammoniacal solution of silver nitrate. The titration is con- 
tinued until a further reduction of the silver solution does not occur, 
which is ascertained by acidifying a few drops of the filtered mixt- 
ure with hydrochloric acid, when in the presence of free silver a 
turbidity referable to silver chloride occurs. Accuracy within nar- 
rower limits than \ c.c. is scarcely possible, as the turbidity refer- 
able to silver chloride can only be recognized within 0.2 to 0.3 c.c. 
According to Baumann, 240 to 245 c.c. of the silver solution repre- 
sent 1 gram of homogentisinic acid. 

Literature. — Bodeker, Annal. d. Chemie u. Pharmakol., 1861, vol. cxvii, p. 
98. Baumann u. Wolkow, Zeit. f. physiol. Chem., 1891, vol. xv, p. 228. Stier, 
Berlin, klin. Woch., 1898, vol. xxxv, p. 185. Embden, Zeit. f. physiol. Chem., 
1893, vol. xvii, p. 182, and vol. xviii, p. 304. Ogden, Zeit. f. physiol. Chem., 1895, 
vol. xx, p. 280. Futcher, N. Y. Med. Jour., 1898, vol. Ixvii, p. 69. Garrod, 
Jour. Physiol., 1899, vol. xxiii, p. 512; and Med.-Chir. Trans. Royal Soc, vol. 
lxxxii, p. 367. E. Meyer, Deutsch. Arch., vol. lxx, Heft 5 u. 6. F. Wittelbach, 
ibid., 1901, vol. lxxi, p. 50. 

Blue Urines. — Blue urines are sometimes seen, the color of which 
is due to indigo formed from urinary indican within the urinary 
passages. Their occurrence can only be regarded as a medical curi- 
osity. One case of this kind is reported by McPhedran and Goldie, 1 
in which after direct extraction of the urine with ether only a faint 
reaction was obtained on further examination, and which probably 
was referable to incomplete previous extraction. Formerly, when 
indigo was employed in the treatment of epilepsy, blue urines were 
frequently seen. At the present time, when methylene blue is occa- 
sionally used in the treatment of malaria and chyluria, this pigment 
is found in the urine. 

Green Urines. — Green urines have also been described; the cause 
of the color, however, has not been ascertained. 

Pigments referable to Drugs. — Certain drugs may also cause changes 
in the normal color of urine, and in doubtful cases inquiry in this 
direction should be made. It has been pointed out that carbolic 
acid, hydroquinone, pyrocatechin, and salol cause the appearance 
of a dark-brown color, and that after the administration of indigo 

1 Transactions Association American Physicians, 1901. 



522 THE URINE 

and methylene blue blue urines are voided. Santonin, rheum, and 
senna color urines a bright yellow, so that they may resemble icteric 
urines. The yellow color in such cases is changed to an intense red 
by the addition of an alkali, and, if ammoniacal fermentation is going 
on at the same time in the bladder, the patient may believe himself 
to be suffering from hematuria. The red color thus produced is due 
to the action of the alkali upon chrysophanic acid. When urines 
containing copaiba are treated with hydrochloric acid a red color 
results, which changes to violet upon the application of heat. During 
the administration of potassium iodide, or the use of iodine in any 
form, a dark mahogany color is obtained when the urine is treated 
with nitric acid. In doubtful cases Stokvis' modification of Jaffe's 
test for indican should be employed, when in the presence of an iodide 
the chloroform assumes a beautiful rose-red color. 

For the detection of other drugs and poisons in the urine the reader 
is referred to special works. 

Ehrlich's Diazo Reaction-. — Under certain pathological conditions, 
and especially in typhoid fever, a chromogen may be present in the 
urine, which, when treated with diazo-benzene-sulphonic acid and 
ammonia, imparts a red color to the urine, varying from eosin 
to a deep garnet red. This reaction, which is generally spoken 
of as Ehrlich's reaction, or the diazo reaction, was at one time re- 
garded as pathognomonic of typhoid fever. Subsequent exami- 
nations, however, have shown that it may also be present in other 
diseases. Michaelis, who has made an exhaustive study of this 
question, divides into four groups the diseases in which the reac- 
tion has been observed. In the first group, comprising diseases of 
the nervous system, chronic diseases of the heart and kidneys, 
malignant tumors, etc., the reaction is rarely seen. When present, 
it usually indicates a secondary infection. The second group in- 
cludes those diseases in which the reaction is almost always present, 
namely, typhoid fever and measles. In the diseases of the third 
group it is often, though not invariably, observed. Under this 
heading are classed scarlet fever, erysipelas, pneumonia, diphtheria, 
pyemia, acute miliary tuberculosis, etc. The fourth group comprises 
pulmonary tuberculosis, and includes acute caseous pneumonia. 

The value of Ehrlich's reaction in typhoid fever was at first overesti- 
mated, but is at present certainly underestimated. I have studied 
this problem with great care, and after many years' experience 
maintain, as I did years ago, that the test is a most important diag- 
nostic aid in the disease in question. As a general rule the reac- 
tion is present as early as the fifth or sixth day, and may persist into 
the third week; it then disappears, but may reappear when a relapse 
occurs. This fact is generally overlooked and should be borne in 
mind in the differental diagnosis from acute tuberculosis. Excepting 
in children, its absence from the fifth to the ninth day usually indicates 



PLATE XIX, 




Diazo-reaetion, as modified by the author. 

rolorinthe lower portion of the test tube may 

n any urine ; the dark carmine ring indicates 

of the reaction in a well-pronounced degree ; 

zone above is intended to indicate the arn- 

has been added. 



CHEMISTRY OF THE URINE 523 

a mild case. This rule, however, is not without exception. When 
the reaction is continuously present after the third week I am inclined 
to suspect acute tuberculosis. It may be present as early as the 
fourth day of the disease. 

In paratyphoid, as in typhoid fever, the reaction is also fairly con- 
stant. 

Of late much attention has been paid to the occurrence of Ehrlich's 
reaction in pulmonary phthisis. As a result of his investigations 
Michaelis concludes that its presence in such cases indicates either 
that the process is very extensive or that it will progress very rapidly, 
and that the prognosis is grave. A cure, be believes, is impossible, 
and improvement, if any, only temporary. Clemens notes that of 
100 cases of phthisis which ended fatally 87 showed the diazo reac- 
tion; Rutimeyer obtained positive results in 85 cases out of 106 which 
died. Of 13 cases of acute tuberculous pneumonia Frankel and Troje 
found a positive reaction in 11. Grundriss states that in his fatal 
cases the reaction was present without exception. Similar results 
have been obtained by Cnopf, See, Goldschmidt, and others. 
Michaelis himself reports that of 111 cases of phthisis which were 
admitted to the Berlin Charite with well-marked reaction 80 died 
in the hospital, 13 were discharged unimproved, 3 were transferred 
to other hospitals, and 15 left improved. In other words, of these 
111 cases a fatal result was known to have occurred in 72 per cent. 
Stadelmann states that of 38 other cases with positive reaction 28 
died in the hospital — i. e.- } about 75 per cent. The subsequent fate 
of the remaining cases was not ascertained; but we may well assume 
that of these at least 50 per cent, died; so that we may formulate the 
general rule that a fatal result may be anticipated in about 85 per 
cent, of all cases of phthisis in which a positive reaction is obtained. 
Michaelis, moreover, suggests that the end may be expected to occur 
within six months from the time at which a persistent Ehrlich reac- 
tion is established. Exceptions occur, but the above is the rule. In 
Koch's institute at Berlin patients presenting the diazo reaction are 
not treated with tuberculin (Brieger). 1 

In tuberculous peritonitis the diazo reaction is found in about 25 
per cent, of all cases. 

As regards the frequency of occurrence of the reaction in diph- 
theria, it appears from the observations of Rivier 2 and others that 
it is decidely uncommon. Of his own 118 cases, and 44 additional 
ones collected from the literature, only 10 gave a positive result; 
and of these, 4 should be eliminated as they occurred in com- 
plicated cases; so that the reaction was absent in about 97 per 
cent. 

1 Discussion on Tuberculosis, Michaelis, Deutsch. med. Woch., 1901, vol. v, p. 
211. 

2 These de Paris, 1898. 



524 THE URINE 

In the scarlatiniform erythema due to serum treatment the 
reaction is absent, while in true scarlatina it is fairly common. 
Including a number of cases collected from the literature Rivier 
found a positive reaction in 41 cases out of 73. He concludes that 
in the differential diagnosis between the two conditions scarlatina 
may be affirmed if the reaction is positive, while if negative there is 
strong presumptive evidence against the disease. 

In measles a positive reaction was obtained in 75 of 85 cases. 

Ruttimeyer obtained the reaction in pulmonary actinomycosis. 

The reaction has been referred to the presence of alloxyproteinic 
acid, 1 but this is denied by Clemens. 

As the preparation of chemically pure, crystalline diazo com- 
pounds is a difficult process, Ehrlich uses sulphanilic acid, which, 
when treated with nitrous acid in a nascent state, gives rise to the 
formation of diazo-benzene-sulphonic acid, as is shown by the 
equations : 

1. NaN0 2 + HC1 = NaCl + HN0 2 . 

/NH 2 /N. 

2. C 6 H 4 ( + HN0 2 = C 6 H 4 ( ^N + 2H 2 0. 

\S0 3 H X S0 3 / 

Para-amino- Diazo-benzene- 

benzene-sulphonic acid. sulphonic acid. 

This is the active principle in the mixture employed. 

Other compounds may, of course, also be used, such as meta-amino- 
benzene-sulphonic acid, ortho- and para-toluidin-sulphonic acid, etc. ; 
but of all these, Ehrlich found the common sulphanilic acid the most 
convenient. Two solutions, which must be kept in separate bottles, 
are employed. The one is a 5 per cent, solution of hydrochloric 
acid, to which sulphanilic acid is added in the proportion of 1 gram 
for every 100 c.c. The other is a 0.5 per cent, solution of sodium 
nitrite. 

The two solutions are mixed in the proportion of 40 to 1 im- 
mediately before using. A few cubic centimeters of urine are then 
treated with an equal volume of the reagent; the mixture is shaken 
and rendered alkaline with ammonium hydrate. This is best 
allowed to flow down the sides of the tube, so as to form a layer 
above the mixture. At the junction of the two fluids a colored ring 
will now be observed. With urines which do not contain the chro- 
mogen this will be a more or less distinct orange, while in its pres- 
ence a red color is obtained. The intensity of this color may vary 
from eosin to a deep garnet red. If the mixture is now agitated 
and the reaction is positive, the foam will likewise be colored red, 
and upon pouring the solution into a porcelain basin containing 

1 Bondzynski u. Panek, Berlin, d. deutsch. chem. Ges., 1903, vol. xxxv, p. 
2951. 



CHEMISTRY OF THE URINE 525 

much water a beautiful salmon color is obtained, even if only traces 
of the chromogen are present. Carried out in this manner no ques- 
tion will arise as to the presence or absence of the reaction. Ehr- 
lich states that on standing a green sediment forms in the alkalinized 
mixture, and he regards this sediment as especially characteristic. 
My experience has been that this becomes manifest only when the 
color reaction is well pronounced, and I am inclined to attach more 
importance to the salmon color obtained upon copious dilution. 
With normal urines this is never obtained, and it can still be 
seen when inspection of the fluid in the test-tube would leave in 
doubt. 

The older method of Ehrlich I have abandoned, as the test just 
described is simpler, and, in my experience, just as reliable. He 
advised the addition of about 50 c.c. of absolute alcohol to 10 c.c. 
of urine, subsequent filtration, and examination of the filtrate, as 
just described. 

Greene states that if 1 part of the sodium nitrite solution is added 
to 100 instead of 40 parts of the sulphanilic acid solution, a positive 
reaction is no longer obtained in cases of croupous pneumonia and 
of pulmonary tuberculosis, while in typhoid fever the reaction occurs 
with the same intensity. 

While in the absence of the chromogen, as I have already stated, 
a more or less pronounced orange color is usually obtained, excep- 
tions have been noted. Ehrlich thus records that in urines contain- 
ing biliary coloring matter an intensely dark, cloudy discoloration 
occurs at times, which upon boiling is changed to a well-marked 
reddish violet. In rare instances of ulcerative endocarditis, hepatic 
abscess, and intermittent fever, and more commonly in pneumonia 
about the time of the crisis, Ehrlich further observed an intense yolk- 
yellow color, before the addition of the ammonia, which becomes 
somewhat lighter after this is added. The reaction is supposedly 
referable to urobilinogen {egg-yellow reaction). 

Of interest is the observation of Burghart, that after the adminis- 
tration of tannic acid, gallic acid, and certain iodine preparations, 
Ehrlich's reaction disappears from the urine. But, as Burghart 
himself suggests, it is likely that this inhibitory effect is not exerted 
upon the diazo-forming substance, but upon the reagents employed. 
Other factors, which may prevent the occurrence of Ehrlich's reac- 
tion, in pulmonary tuberculosis at least, are the occurrence of renal 
complications (albuminuria). Naphthalin, after its administration 
by the mouth, according to my experience may cause a reaction, the 
color of which corresponds exactly to that of the diazo reaction. 

Other observers have noted a similar reaction after the adminis- 
tration of opium (morphine, heroine), alcohol in large amount, phenol, 
cresol, creosote, and guaiacol. Golden, on the other hand, denies its 
occurrence after the use of some of the substances mentioned. 



526 THE URINE 

Literature. — Ehrlich, Zeit. f. klin. Med., 1882, vol. v, p. 285; Charit. Annal., 
1883, vol. viii, p. 28, and 1886, vol. xi. p. 139. Goldschmidt, Munch, med. 
Woch., 1886, vol. xxxiii, p. 35. Riitimeyer, Corresp. Blatt. f. Schweizer Aerzte, 
1890, vol. xxvi. Greene, Med. Record, Nov. 14, 1896. C. E. Simon, Johns 
Hopkins Hosp. Bull., 1890. J. Friedenwald, N. Y. Med. Jour., 1893. M. 
Michaelis, Berlin, klin. Woch., 1900, p. 274; and Deutsch. med. Woch., 1899, p. 
156. J. R. Arneill, Amer. Jour. Med. Sci., 1900, p. 296. 

Ehrlich's Dimethylaminobenzaldehdye Reaction. — Ehrlich has shown 
that under various pathological conditions a fine cherry-red color 
develops on shaking a specimen of urine with a few drops of dimethyl- 
aminobenzaldehyde in acid solution, and that the resulting pigment 
can be in part extracted with chloroform, and almost entirely so with 
epi- or dichlorhydrin. With normal urines a similar reaction can be 
obtained, but it is much less intense, and if done at ordinary tempera- 
tures a distinct red color does not develop. On heating, however, 
it appears, and can likewise be extracted with epichlorhydrin. The 
reaction, according to O. Neubauer, is due to urobilinogen. 

As regards the occurrence of the reaction in disease I can summarize 
my results as follows : (1) A direct reaction, of pathological grade, does 
not occur in health. (2) A positive reaction is most commonly ob- 
tained in cases of tuberculosis. (3) It may also be seen in non-tuber- 
culous cases, both febrile and non-febrile. (4) It is not dependent 
upon the presence of the body which gives rise to the diazo reaction. 
(5) For its production elevation of temperature, gastro-intestinal dis- 
turbances, and cyanosis are not essential. (6) Common to all cases 
seems to be an increased katabolism of the tissue albumins. 

My positive results include cases of pulmonary tuberculosis, 
tuberculosis of the hip-joint, pneumonia, typhoid fever, appendi- 
citis, embarras gastrique, icterus, malignant endocarditis, empyema, 
esophageal carcinoma, and a remarkable instance of traumatic neu- 
rosis, in which a loss of weight of from sixty to seventy-five pounds 
had occurred. 

My list of negative cases, on the other hand, includes, first of 
all, a large number of normal or supposedly normal individuals; in 
addition, cases of normal labor, neurasthenia, hysteria, diabetes, 
aortic aneurysm, myelogenous leukemia, lymphatic leukemia, acute 
nephritis (scarlatinal), simple diarrhea, morphinism, valvular dis- 
ease, phthisis (stationary), diphtheria (before and after the use of 
antitoxin), typhoid fever, cases of abortion, appendicitis, influenza, 
chronic nephritis, cystitis, pyelitis (calculous), measles, tuberculosis 
of the hip-joint, cystic kidney, carcinoma of the kidney, tonsillitis, 
acute and chronic bronchitis, pneumonia, icterus, tuberculous perito- 
nitis, general erythema; varicocele; following various operations, 
such as nephrorrhaphy, removal of pus tubes, operations for vesico- 
vaginal fistula, fistula in ano, and suspension of the uterus. Exami- 
nation of a urine containing cystin and diamins was also negative. 
A comparison of the negative with the positive cases will show at 



CHEMISTRY OF THE URINE 527 

once that not all cases of pulmonary tuberculosis, tuberculous hip- 
joint disease, pneumonia, typhoid fever, appendicitis, and icterus 
give a positive result. So far as tuberculosis is concerned, however, 
it appears that the reaction is more likely to occur in the actively 
progressive cases than in those which are more or less stationary. 
It was also noted that the positive cases almost all gave a positive 
diazo reaction, while in the negative cases this was not obtained. 
Exceptions, however, may also occur. 

In my personal examinations I employed a 2 per cent, solution 
of dimethylparaminobenzaldehyde in equal parts of water and con- 
centrated hydrochloric acid. A few cubic centimeters of urine in a 
test-tube are treated with from 5 to 10 drops of the reagent; the 
mixture is set aside or agitated for a few minutes and the color then 
noted. Normal urines usually turn a greenish yellow, or the normal 
color merely becomes intensified. At times a dark-amber color 
develops, though this is less common in health, unless the urine is 
brought to the boil before the reagent is added. In this way it is a 
common experience to meet with moderate or dark-amber tints. 
With these reactions, however, I have not occupied myself, and, 
like Clemens and Koziczkowsky, I have only noted the reaction as 
positive when a distinct cherry-red color developed, either immediately 
on adding the reagent or after agitation or standing. 

Literature. — Ehrlich, med. Woch., 1901, No. 15. Clemens, Deutsch. Arch., 
1901, vol. lxxi, p. 168. Koziczkowsky, Berl. med. Woch., 1902, vol. xxxix, 
No. 44. Simon, Amer. Jour. Med. Sci., 1903, vol. cxxvi, p. 471. 



Acetone. 

The amount of acetone which may be found in the urine under 
normal conditions varies between 0.008 and 0.027 gram, and is 
greatly influenced by the character of the diet. Whenever the car- 
bohydrates are withdrawn the quantity rapidly increases and reaches 
its maximum about the seventh or eighth day. At this time from 
200 to 700 mgrms. may be eliminated in the twenty-four hours. 
If, then, carbohydrates are again added to the diet, the acetonuria 
soon disappears. This result is not reached, however, if fats are sub- 
stituted for the carbohydrates. The acetonuria is greatest when but 
little albuminous food and no carbohydrates at all are ingested, and 
during starvation the same amounts are essentially found. Increased 
amounts are found in fevers, the various cachexias, in conditions 
associated with inanition, etc. 1 The source of the acetone in these 
cases was formerly sought in the increased albuminous destruction, 

1 v. Jaksch, Ueber Acetonurie u. Diaceturie, Hirschwald, Berlin, 1885. Rosen- 
feld, Centralbl. f. inn. Med., 1895, vol. xv. Waldvogel, " Zur Lehre von der 
Acetonurie," Zeit. f. klin. Med., vol. xxxviii, p. 506. 



528 THE URINE 

but according to more recent research it appears that in some manner 
the fat metabolism is involved and. that the acetonuria is the result. 

Most important is the diabetic form of acetonuria. It may be 
stated, as a general rule, that the diagnosis of diabetes mellitus is 
justifiable whenever sugar and notable quantities of acetone are 
found in the urine. The amount of acetone, moreover, stands in a 
direct relation to the intensity of the disease, the maximum excretion 
being usually observed toward the fatal end. 1 Whether or not this 
form of acetonuria can always be explained upon the basis given 
above remains an open question. There can be no doubt, however, 
that the threatening symptoms which are so commonly associated 
with a greatly increased elimination of acetone will often disappear, 
at least temporarily, if carbohydrates are administered in large 
amounts. It is certain, moreover, that diabetic coma is more apt to 
occur when the old-fashioned plan of excluding carbohydrates 
entirely from the dietary of diabetic patients is adopted. Hirschfeld 2 
suggests that in every case of diabetes the excretion of acetone be 
carefully followed, and that large amounts of carbohydrates be ad- 
ministered whenever the acetonuria approaches a dangerous extent. 

Of the febrile diseases in which acetonuria has been observed 
may be mentioned typhoid fever, pneumonia, scarlatina, measles, 
acute miliary tuberculosis, acute articular rheumatism, and septi- 
cemia. In those of short duration, on the other hand, even if the 
fever is high, as in acute tonsillitis, intermittent fever, the hectic 
fever of phthisis, etc., an increased elimination of acetone is rarely 
observed. In the continued fevers the acetonuria is largely referable 
to the character of the diet, as carbohydrates are usually excluded 
entirely, and I have repeatedly observed that a return to the normal 
occurred as soon as sugar was administered in amounts varying from 
50 to 100 grams. 

In certain nervous and mental diseases, as in general paresis, 
melancholia, following epileptic seizures, and in tabes, acetonuria is 
frequently observed. During the second stage of general paresis 
increased amounts are indeed quite constantly found, but Hirschfeld 
is probably correct in stating that the psychotic form of acetonuria 
is largely referable to improper feeding. 

A notable degree of acetonuria has been observed in connection 
with the pernicious vomiting of pregnancy, 3 and in eclampsia (Bag- 
inski). A certain amount of acetone occurs normally during the first 
two days of the puerperal period, but usually disappears by the third 
day. 

1 v. Jaksch, Zeit. f. klin. Med., 1885, vol. x, p. 362. Lorenz, ibid., 1891, vol. 
xix, p. 19. 

2 Beobachtungen liber d. Acetonurie u. das Coma diabeticum, Zeit. f. klin. 
Med., vol. xxviii, p. 176, and vol. xxxi, p. 212. 

3 H. Baldwin, Amer. Jour., Oct. 1905, p. 649. 



CHEMISTRY OF THE URINE 529 

According to Vicarelli 1 acetonuria occurring in the course of preg- 
nancy is evidence of the death of the fetus. This is possibly the 
rule, but exceptions have been observed. 

In the primary diseases of the stomach, and notably in carcinoma, 
acetonuria is frequently observed, and it is possible that the acetone 
in these cases is, to some extent at least, formed in that organ directly 
from the proteids ingested. The facts that in carcinoma acetone may 
be observed at a time when marked loss of flesh has not as yet occurred, 
and that larger amounts of acetone may be found in the stomach 
than in the urine, are certainly in favor of this view. 2 

An enterogenic form of acetonuria has further been described, and 
it has been urged that in these cases the acetone is referable to the 
formation of unusually large amounts of fatty acids. Acetonuria 
of this type is also observed following the ingestion of fatty acids 
as such (alimentary form). 3 

Acetonuria has further been observed early in the course of acute 
phosphorus poisoning, and may persist throughout, apparently with- 
out being an index of the severity of the case. 

After chloroform narcosis the condition is also not uncommon. 

Tests for Acetone. Legal's Test. 4 — This test may be applied to 
the freshly voided urine, but is not conclusive. Several cubic centi- 
meters of urine are treated with a few drops of a strong solution of 
sodium nitroprusside and sodium hydrate; the mixture assumes a 
red color, which rapidly disappears, and in the presence of acetone 
is replaced by a purple or violet red when acetic acid is added. As 
a rule, it is better to distil the urine (500 to 1000 c.c.) after the addi- 
tion of a little phosphoric acid (1 gram pro liter), and to employ the 
first 10 to 30 c.c. of the distillate for one or more of the following 
tests. 

Lieben's Test. 5 — A few cubic centimeters of the distillate are 
rendered strongly alkaline with caustic soda solution and treated 
with several drops of a dilute solution of iodopotassic iodide, when 
in the presence even of traces of acetone a precipitation of iodoform 
in crystalline form occurs. This may be recognized by its odor 
when the solution is heated, as also by the form of the crystals, which 
occur as hexagonal or stellate platelets. If traces of acetone only are 
present it is necessary to let the solution stand for a number of hours 
before examining. 

Alcohol and acetic aldehyde give the same reaction. For this rea- 

1 Prager med. Woch., 1893, Bd. xxxiii und xxxv; also Knapp, Centralbl. f. 
Gynak, 1897. 
* 2 H. Lorenz, loc. cit. 

3 Waldvogel u. Hagenberg, "Ueber alimentare Acetonurie," Zeit. f. klin. Med., 
1900, vol. xiii, p. 443. 

4 Le Nobel, Arch. f. exper. Path. u. Pharmakol., 1884, vol. xviii, p. 9. 

5 Taniguti u. Salkowski, Zeit. f. physiol. Chem., 1890, vol. xiv, p. 476. 

34 



530 THE URINE 

son Dunning' s modification 1 is sometimes to be preferred, although it 
is not as delicate. To this end a small amount of Lugol's solution is 
added to the distillate and a sufficient amount of ammonia to produce 
a black precipitate (nitrogen iodide). This disappears on standing 
and in the presence of acetone is replaced by iodoform. 

Gunning's test, like that of Legal, may be tried with the native urine 
first. 

Frommer's Test. 2 — This test also may be applied directly to the 
urine, and is said to indicate the presence of 0.000001 acetone in 8 
c.c. of water. It does not react with diacetic acid. 

About 10 c.c. of urine are treated with about 1 gram of caustic 
soda in substance and — without waiting for the dissolution of the soda 
to occur — with 10 to 12 drops of an alcoholic solution of salicylic 
aldehyde (1 gram to 10 c.c. of absolute alcohol). The mixture 
is heated to 70° C. In the presence of acetone a marked purple-red 
color results at the zone of contact with the alkali. 

If the alkali is added in solution the fluid first becomes yellow, 
later reddish, then purplish red, and finally dark carmine red. The 
color change occurs more rapidly by heating. 

Denniges' Test (as Modified by Oppenheimer). 3 — The reagent is 
prepared as follows: 20 grams of concentrated sulphuric acid are 
poured into 100 c.c. of distilled water, when 5 grams of freshly pre- 
pared yellow mercuric oxide are added. The mixture is allowed to 
stand for twenty-four hours and is then ready for use. 

This reagent is added to about 3 c.c. of urine, drop by drop, until 
the precipitate which is thus formed no longer disappears on stirring. 
When this point is reached a few more drops are added. After two 
or three minutes the precipitate is filtered off. The clear filtrate is 
further treated with about 2 c.c. of the reagent and 3 to 4 c.c. of a 
30 per cent, solution of sulphuric acid, and boiled for a minute or 
two, or, still better, placed in a vessel with boiling water. In the 
presence of an abundant amount of acetone a copious white precipi- 
tate forms immediately; while in the presence of traces only (less 
than 1 to 50,000), a slight cloud develops on standing for several 
minutes. The precipitate is almost entirely soluble in an excess of 
hydrochloric acid. 

If albumin is present, the urine becomes turbid at once when the 
reagent is added. In that case the test is continued as described, 
attention being directed to the coarser precipitate which occurs later. 
To such urines large amounts of the reagent must be added, the idea 
being to precipitate everything that can be precipitated with the 
reagent, before heating. 

Oppenheimer claims that the test is as delicate as that of Lieben, 

1 Jour, de pharmacol. et de chim., 1881, vol. iv, p. 30. 

2 Berlin, klin. Woch., Aug. 7, 1905, p. 1008. 

3 Ibid., 1899, p. 828. 



CHEMISTRY OF THE URINE 531 

viz., giving a well-pronounced reaction with a dilution of 1 to 20,000, 
and being still discernible with a dilution of 1 to 60,000. As diacetic 
acid yields acetone when treated with mineral acids, a positive result 
is always obtained when this is present. But as diacetic acid is 
usually found only in association with acetone, this fact does not 
lessen the value of the test, and is an error, moreover, which is common 
to the other tests as well. 

Quantitative Estimation of Acetone. — For the purpose of estimat- 
ing the amount of acetone the method of Messinger, as modified by 
Huppert, is now employed, and is greatly to be preferred to the older 
procedure of v. Jaksch. 1 

Principle. — The method is based upon the observation of Lieben 
that acetone gives rise to the formation of iodoform when treated 
with iodine in an alkaline solution. If then a solution of acetone is 
treated with a known amount of iodine, it is a simple matter to 
determine the quantity present by retitrating the iodine which was 
not used in the formation of iodoform. 

Solutions required: 

1. Acetic acid (50 per cent, solution). 

2. Sulphuric acid (12 per cent, solution). 

3. Sodium hydrate solution (50 per cent.). 

4. A decinormal solution of iodine. 

5. A decinormal solution of sodium thiosulphate. 

6. Starch solution (see Boas' method of estimating lactic acid). 
Preparation of the solutions: 

1. The decinormal solution of iodine is prepared as described 
elsewhere (see Boas' method of estimating lactic acid). 

2. As the molecular weight of sodium thiosulphate — Na 2 S 2 3 .- 
5H 2 — is 248, a decinormal solution of the salt would contain 24.8 
grams to the liter. This quantity is dissolved in about 950 c.c. 
of distilled water and brought to the proper strength by titration 
with a decinormal solution of iodine. As 1 c.c. of the thiosulphate 
solution should correspond to 1 c.c. of the iodine solution, the neces- 
sary amount of water which must be added to the former is then 
determined. 

Method. — 100 c.c. of urine, or less if much acetone is present, 
as determined by Legal's test, are treated with 2 c.c. of the 
acetic acid solution and distilled until seven-eighths of the total 
amount have passed over. The distillate is received in a retort 
which is connected with a bulb tube containing water. As soon 
as seven-eighths of the urine have distilled over, a small amount 
of the distillate of the remainder is tested for acetone according 
to Lieben's method. Should a positive reaction be obtained, it 
will be necessary either to repeat the entire process with less urine, 

1 See Neubauer u. Vogel, Analyse des Harris, 9th ed., p. 470. 



532 THE URINE 

diluted to about 200 c.c., or to add about 100 c.c. of water to the 
residue and to distil until all the acetone has passed over. The 
distillate is then treated with 1 c.c. of the sulphuric acid and redis- 
tilled. The addition of the acetic acid and of the sulphuric acid, 
respectively, serves the purpose of holding back phenol and am- 
monia. Should the first distillate contain nitrous acid, moreover, 
which is recognized by the addition of a little starch paste contain- 
ing a trace of potassium iodide, when the solution turns blue, the 
acid is removed by adding a little urea. The second distillate is 
received in a bottle provided with a well-ground glass stopper, and 
holding about 1 liter. The distillate is then treated with a carefully 
measured quantity of the one-tenth normal solution of iodine — ■ 
about 10 c.c. for 100 c.c. of urine — and sodium hydrate solution 
until the iodoform separates out. To this end a slight excess of the 
solution must be added. Should ammonia be present, a blackish 
cloud will be observed at the zone of contact of the sodium hydrate 
and the iodine solution, and it will be necessary to repeat the entire 
process. The bottle is closed and shaken for about one minute. 
The solution is then acidified with concentrated hydrochloric acid, 
when the mixture assumes a brown color if iodine is present in 
excess. If this does not occur more of the iodine solution must 
be added and the process repeated until an excess is present. The 
excess is then retitrated with the thiosulphate solution until the fluid 
presents a faint-yellow color. A few cubic centimeters of starch 
solution are now added, and the titration continued until the last 
trace of blue has disappeared. The number of cubic centimeters 
employed in the titration is finally deducted from the total amount 
of the iodine solution added, and the result multiplied by 0.976. 
The figure thus obtained indicates the amount of acetone contained 
in the 100 c.c. of urine, in mgrms., as 1 c.c. of the thiosulphate solu- 
tion is equivalent to 1 c.c. of the iodine solution, or to 0.967 mgrm. 
of acetone. 

Diacetic Acid. 

The occurrence of diacetic acid in the urine must always be 
regarded as abnormal. Its pathological significance is identical with 
that of acetonuria. It is met with especially in diabetes, in various 
digestive diseases, and in febrile diseases. In the continued fevers 
of childhood it is almost constantly present. H. Baldwin noted its 
presence in a case of pernicious vomiting of pregnancy. 

Gerhardt's Test. — To demonstrate the presence of diacetic acid a 
few cubic centimeters of urine are treated with a strong solution of 
ferric chloride drop by drop. A precipitate of phosphates is filtered 
off, when more of the iron solution is added to the filtrate. If a 
Bordeaux red color appears, this may be due to diacetic acid. To 



CHEMISTRY OF THE URINE 533 

make sure another portion of urine is boiled and similarly treated. 
As diacetic acid is decomposed on boiling no reaction at all or only a 
faint reddish color should be obtained. As further proof a third 
portion of urine is acidified with sulphuric acid and extracted with 
ether. The diacetic acid is thus isolated. A positive reaction, when 
the ethereal extract is shaken with ferric chloride will indicate the 
presence of diacetic acid. The color disappears on standing for 
twenty-four to forty-eight hours. A similar reaction is obtained with 
salicylic acid, antipyrine, sodium acetate, and other aromatic com- 
pounds, but the color persists for days. Sulphocyanides like diacetic 
acid will pass into the ethereal extract, but the color does not disap- 
pear on standing. 

Arnold's Test (Modified by Lipliawski). — Two solutions are employed, 
viz., a 1 per cent, solution of para-amido-aceto-phenone and a 1 per 
cent, solution of potassium nitrite. 6 c.c. of the first solution and 
3 c.c. of the second are added to an equal volume of urine, together 
with a drop of concentrated ammonia. The mixture is shaken until 
it assumes a brick-red color. From 10 drops to 2 c.c, according to 
the amount of diacetic acid present, are treated with 15 to 20 c.c. of 
concentrated hydrochloric acid (sp. gr. 1.19), 3 c.c. of chloroform, 
and 2 to 4 drops of an aqueous solution of ferric chloride. The tube 
is closed with a cork and gently agitated (so as to avoid emulsifica- 
tion), when after one-half to one minute a beautiful and very charac- 
teristic violet tinge results if diacetic acid is present. In its absence 
the color is yellowish or slightly reddish. The violet color persists 
for a long time. Bilirubin, salicylic acid, phenacetin, antipyrine, 
phenol, and other drugs are without disturbing influence upon the 
reaction. Highly colored urines should first be filtered through ani- 
mal charcoal. 

Allard states that both Arnold's test and that of Lipliawski give 
a positive result also with acetone, when this is present to the extent 
of more than 1 per cent. 

Literature. — v. Jaksch, Ueber Acetonurie u. Diaceturie, loc. 'tit. Ibid., 
Zeit. f. Heilk., 1882, vol. iii, p. 34. Schrack, Jahrbuch f. Kinderheilk., 1889, 
vol. xxix, p. 411. v. Arnold, Wien. klin. Woch., 1899, p. 541. 



Oxybutyria Acid. 

The fact that in some cases of diabetes an excessive elimination 
of ammonia was observed led to the belief that there must be pres- 
ent an unknown acid; this was shown to be /9-oxybutyric acid. The 
occurrence of this acid in the urine of diabetic patients is of great 
clinical interest, not only from the standpoint of diagnosis, but also 
of prognosis and treatment. Its presence may always be regarded 
as indicating a severe type of the disease, and when associated with 



534 THE URINE 

marked acetonuria and diaceturia as indicating the possible occurrence 
of coma. 

According to Herter, the condition of diabetic coma is preceded by a 
period of days, weeks, or months, in which there is a large excretion 
of /3-oxybutyric acid (20 grams or more in twenty-four hours), and 
in which the nitrogen in the form of ammonia is largely increased. 
The same writer states that patients whose urines show or have 
shown a large excretion of organic acids are in danger of devel- 
oping diabetic coma; but the nitrogen of ammonia may temporarily 
rise as high as 16 per cent., and yet coma may be delayed for more 
than seven months. The persistent excretion of more than 25 
grams of /9-oxybutyric acid indicates impending coma. Impor- 
tant also is the observation that while as a general rule the appear- 
ance of large amounts of organic acids is associated with the presence 
of much sugar, a constant relation between the two does not exist. 
There may thus be much sugar and little or no acid in the urine, or 
there may be much acid and little sugar. 

Besides diabetes, the substance may be met with in scarlatina, 
measles, scurvy, and in starving insane patients. 

The presence of oxybutyric acid may be inferred in diabetic urines 
if after fermentation a rotation of the plane of polarization to the 
left is observed. Albumin, if present, must first be removed. 

Quantitative Estimation according to Darmstaedter. — This 
method is based on the decomposition of the /9-oxybutyric acid with the 
formation of a-crotonic acid and the estimation of the latter. This 
decomposition takes place according to the equation : 

CH 3 .CHOH.CH 2 .COOH = CH 3 .CH.CH.COOH + H 2 0. 

/3-oxybutyric acid. Crotonic acid. 

100 c.c. of urine are rendered feebly alkaline with sodium carbo- 
nate and evaporated on a water bath almost to dryness. With 
the aid of 150 to 200 c.c. of sulphuric acid (50 to 55 per cent.) the 
residue is transferred to a liter flask, which is closed with a doubly 
perforated stopper. Through the one aperture a drip-tube passes, 
while a bent glass tube passes through the other to a condenser. 
Heat is applied, at first mildly, so as to avoid foaming; then vigorously. 
Water is allowed to enter through the drip-tube as fast as the dis- 
tillate passes over. The distillation is interrupted when from 300 
to 350 c.c. have been obtained, which usually takes from two to two 
and one-half hours. The distillate is extracted two or three times 
with ether. The ether is distilled off, the residue heated for a few 
minutes on a sand bath to 160° C. in order to drive off any fatty acids 
that may be present, and then dissolved on cooling with 50 c.c. of 
water. The solution is filtered and the filter washed with a little 
water. The aqueous solution of the crotonic acid is now titrated 
with a decinormal sodium hydrate solution, using phenolphthalein as 



CHEMISTRY OF THE URINE 535 

an indicator. 1 c.c. of the soda solution corresponds to 0.0086 
gram of crotonic acid. The corresponding amount of oxybutyric 
acid is obtained by multiplying by 1.21. Sugar does not interfere 
with the process. 

If it is only desired to prove the presence of oxybutyric acid in 
the urine, this method can also be conveniently employed. The cro- 
tonic acid is obtained from the ethereal extract, and recognized by 
its melting point, 72° C. If necessary, it can be purified by solu- 
tion in water and reextraction with a small amount of ether and 
subsequent evaporation, viz., distillation of the ether. 

Literature. — v. Jaksch, Ueber Acetonurie u. Diaceturie, loc. cit. H. Wolpe, 
Arch. f. exper. Path. u. Pharmakol., 1886, vol. xxi, p. 131. Herter, "The Acid 
Intoxication of Diabetes in its Relation to Prognosis," Jour, of Exper. Med., 
1901, vol. v, p. 617. E. Darmstaedter, Zeit. f. phys. Chem., 1903, vol. xxxvii, 
p. 355. 

Crotonic Acid. 

As has just been shown, crotonic acid is a derivative of oxybu- 
tyric acid. Its presence in the urine as such has not as yet been 
established, and it is likely that statements to the contrary are based 
upon findings of the acid in the distillate, especially when the dis- 
tillation has been carried on after the addition of sulphuric acid to 
■the urine. But even in the absence of a free acid a small amount 
of crotonic acid results from oxybutyric acid on boiling. 



Lactic Acid. 

Sarcolactic acid is normally absent from the urine, but is met 
with in pathological conditions, and particularly in hepatic diseases, 
as the liver is normally concerned in the decomposition of lactic 
acid and of the lactates that have been ingested with the food. As 
has been pointed out, moreover, there is evidence to show that 
a portion of the nitrogen eliminated from the body reaches the 
liver as ammonium lactate, and is here transformed into urea. 
As a consequence, lactic acid appears in the urine whenever, as 
in phosphorus poisoning, acute yellow atrophy, etc., extensive 
destruction of the hepatic parenchyma occurs, and the formation 
of urea is consequently impaired. In such cases the elimination of 
lactic acid is associated with an increased excretion of ammonia. The 
same will occur when, owing to insufficient oxygenation of the blood, 
the power of oxidation on the part of the liver is interfered with. 
We accordingly find lactic acid in the urine in the chronic anemias, 
in cases of poisoning with carbon monoxide, in association with the 
various forms of circulatory and respiratory dyspnea, in cases of 



536 THE URINE 

epilepsy immediately after the attack, following excessive muscular 
exercise, as in soldiers after forced marches, etc. 

In order to test for lactic acid, the urine is evaporated on a water 
bath to a thick syrup and extracted with 95 per cent, alcohol. This 
is decanted off after twenty-four hours, evaporated to a syrup, acidi- 
fied with dilute sulphuric acid, and extracted with ether, so long as 
this presents an acid reaction. The ether is then distilled off and 
the residue dissolved in water. This solution is treated with a few 
drops of a solution of basic lead acetate, filtered, the excess of lead 
removed by means of hydrogen sulphide, and the filtrate evaporated 
to dryness on a water bath, when the lactic acid will remain behind 
as a slightly yellowish syrup. This is then dissolved in a little 
water, the solution is saturated with zinc carbonate, and boiled. 
Zinc lactate will separate out upon evaporation, especially if a little 
alcohol is added, and may be recognized by the form of its crystals, 
viz., small prisms. These crystals are levorotatory, soluble in alco- 
hol (1 to 1100), and contain two molecules of water of crystallization, 
which is lost at 105° C, so that the loss of weight after heating to 
this temperature must correspond to 12.9 per cent. 

Literature. — O. Minkowski, "Ueber den Einnuss d. Leberextirpation auf d. 
Stoffwechsel," Arch. f. exper. Path. u. Pharmakol., vol. xxi, p. 41; and "Ueber 
Ursache d. Milchsaureausscheidung nach Leberextirpation," ibid., vol. xxxi, p. 
214. G. Colosanti u. R. Moscatelli, " Ueber d. Milchsauregehalt d. menschlichen 
Harns," ibid., vol. xxvii, p. 158. Jnouye and Saiki, " Lactic Acid after Epileptic 
Attacks," Zeit. f. physiol. Chem., 1903, vol. xxxvii, p. 203. 



Oxyamygdalic Acid. 

Schultzen and Riess 1 discovered an acid in the urine of patients 
who had died from acute yellow atrophy to which they gave the 
formula C 8 H 8 4 . They regard it as oxyamygdalic acid and suppose 
it to be derived from tyrosin, which was also found, according to 
the equation: 

C 9 H n N0 3 + 30 = C0 2 + NH 3 + C 3 H 8 4 . 

Very curiously it was not found in cases of phosphorus poisoning, 
but only in acute yellow atrophy. As in this disease there is coin- 
cidently with the rapid parenchymatous destruction much extrav- 
asation of blood, Nencki has suggested that the acid in question 
may possibly be derived from blood pigment, especially as Kuster 
obtained from hematoporphyrin an acid which has the formula 
C 8 H 8 5 , and which thus only differs from the product of Schultzen 
and Riess by a plus of one atom of oxygen. 

1 Annalen d. Charit. Krankenhauses zu Berlin, 1869, vol. xv. 



CHEMISTRY OF THE URINE 537 



Volatile Fatty Acids. 

The term lipaciduria is applied to the elimination of volatile 
fatty acids in the urine. This occurs under normal conditions, but 
may be much more marked in disease. With an ordinary diet the 
degree of lipaciduria corresponds to from 50 to 80 c.c. -^ normal 
sulphuric acid. In febrile conditions, according to v. Jaksch and 
Rokitansky, there is an increase, which runs parallel to the height 
of the temperature. Rosenfeld, however, has shown that this is, 
strictly speaking, not correct, and that an increase is only observed 
in those febrile states in which resorption of breaking-down albu- 
minous material is taking place, as in cases of tonsillar abscess, 
septic diphtheria, putrid bronchitis, and empyema, and in general in 
association with all suppurative processes and hemorrhages within 
the body. Especially high values are found during convalescence 
from pneumonia, during the first days following crisis. This is no 
doubt owing to a resorption of the exudate, and is associated with 
an increased elimination of nitrogen. Immediately before the crisis 
it is common to meet with very low values — 20 c.c. — as compared 
with 100 to 240 c.c. during convalescence. These observations, as 
Rosenfeld has pointed out, may be of marked value in the diagnosis 
of obscure accumulations of pus. 

A marked decrease in the amount of fatty acids is noted in uncom- 
plicated cases of erysipelas and scarlatina (30 to 50 c.c), in measles, 
diphtheria, and, as I have already indicated, in pneumonia preceding 
active resorption of the exudate (20 to 40 c.c). 

According to some observers, the amount of fatty acids in the 
urine may be regarded as an index of the degree of carbohydrate fer- 
mentation in the intestinal tract. Under normal conditions this may be 
the case, but in disease the question is probably more complicated. 

The acids in question are formic acid, acetic acid, butyric acid, 
and propionic acid. They may be isolated as described in the 
chapter on the Feces. 

For their quantitative estimation it will suffice to distil a given 
volume of urine with sulphuric acid and to titrate the distillate 
with j^ normal sodium hydrate solution. The results are expressed 
in terms of the corresponding number of c.c. of -^ normal sulphuric 
acid. 250 c.c of the urine, which must be fresh or preserved with 
chloroform, are distilled with 50 c.c of dilute sulphuric acid until 
200 c.c have passed over. The residue is diluted with 200 c.c. of 
water and the distillation continued as before. In this manner 
the danger that some hydrochloric acid may pass over is avoided, 
but it is well to make sure of this by testing with silver nitrate. 

The method is exact; traces of benzoic acid are included, but in 
man these can be neglected. 



538 THE UBINE 

Literature. — v. Jaksch, Zeit. f. klin. Med., 1886, vol. xi, p. 307; and Zeit. f. 
physiol. Chem., 1886, vol. x, p. 536. 

Blumenthal mentions a case of catarrhal jaundice in which at a 
time when bile again flowed into the intestine a high degree of lip- 
aciduria occurred, viz., up to 385.2 c.c. T \ acid in lieu of the normal 
50 to 80 c.c. 

Amino-acids. 

Tyrosin, leucin, and glycocoll have long been known to occur in 
the urine in acute yellow atrophy and phosphorus poisoning, but 
aside from these conditions nothing further was known of the occur- 
rence of amino-acids under other pathological conditions (barring 
cystinuria). Within recent years, however, and with more exact 
methods it has been possible to show that bodies of this order may 
occur under the most diverse conditions. Phenylalanine alanin, and 
arginin have been found in phosphorus poisoning, besides tyrosin, 
leucin, and glycocoll. 1 Glycocoll indeed, according to a recent 
announcement by v. Noorden, is a normal constituent of the urine and 
may amount to 1 per cent, of 'the total nitrogen output. (This is in 
marked contrast to the statement of Ignatowski 2 that normal human 
urine only contains traces of amino-acids, at best, and that even after 
the subcutaneous injection of 6 grams of glycocoll none is demon- 
strable.) 

Abderhalden 3 found tyrosin in a patient dying with pneumonia, 
who had been suffering from arteriosclerosis, myocarditis, and dia- 
betes. In a second case of diabetes he likewise found tyrosin and 
obtained a marked Millon reaction. In a third case with coma 
tyrosin was present also during the attack, but absent in the interim. 
In a case of severe hepatic cirrhosis a marked ,9-naphthalin sulpho- 
chloride reaction occurred, but it was impossible to isolate amino-acids 
in pure form. The same observer also obtained tyrosin in a case of 
severe icterus, referable to complete occlusion by the common duct, 
and in a patient who had undergone prolonged narcosis; both urines 
gave a marked Millon reaction. 4 Ignatowski found glycocoll con- 
stantly in the urine of 7 gouty patients; in 3 of these also other 
amino-acids, probably leucin and aspartic acid. In pneumonia, 
especially about the time of the crisis and in leukemia he likewise 
obtained positive results. 

Voegtlin and Barker note the occurrence of a distinct Millon reac- 
tion in the urine following the injection of tuberculin for diagnostic 
purposes. 

1 Wohlgemuth, Zeit. f. phys. Chem., 1905, vol. xliv. Abderhalden and Bergell, 
ibid., 1903, vol. xxxix; Abderhalden and L. F. Barker, ibid., 1904, vol. xlii. 

2 Zeit. f. physiol. Chem., 1904, vol. xlii, p. 400. 

3 Ibid., 1905, vol. xliv, p. 50. 

4 Ibid., vol. xlv, p. 468. 



CHEMISTRY OF THE URINE 539 

In this connection the observations of Herter and Wakeman 1 and 
Baldwin 2 are of special interest. Using the method of Magnus- 
Levy 3 of balancing the total bases against the total known acids, they 
found that in certain conditions, notably dilatation of the stomach, 
rheumatoid arthritis, and cirrhosis of the liver, there was a marked 
excess of bases over known acid equivalents. This leads to the 
inference that in the diseases mentioned there must have been pres- 
ent some other organic acid. Magnus-Levy had in this manner pre- 
viously established the presence of such acids in starvation, in intesti- 
nal disturbances, phosphorus poisoning, acute yellow atrophy, and 
fever. 

I append a few of Baldwin's results: 

Apparent Excess of Acids over Bases. 

Average of 10 normal urines 0.2943 

in diabetes mellitus 2 . 96 

in rheumatoid arthritis (active stage) . . . . . 7847 

" " " " , . . . . 0.5598 

" " " ..... 0.6983 

" " " " " 0.6456 

"(case 16) " 0.8377 

Fat. — Under strictly normal conditions the urine contains no fat, 
while variable amounts may be found in disease. When present in 
large quantities, so that it is possible to recognize it with the naked eye, 
the condition is termed lipuria. Such cases, however, are rare, and 
the diagnosis should only be made when it is possible to exclude 
accidental contamination. Smaller quantities, recognizable only with 
the microscope, are much more common, and are indeed quite con- 
stantly observed whenever fatty degeneration of the renal epithelial 
cells, of pus corpuscles, or of tumor particles is taking place in the 
urinary tract. The fat droplets may then be found floating in the 
urine or attached to or embedded in any morphological elements that 
may be present. Lipuria may also occur when abnormally large 
quantities of fat are circulating in the blood. It is thus observed 
after the administration of cod-liver oil in large quantities, following 
oil inunctions, in cases of fracture of the long bones with extensive 
destruction of the bone-marrow, in cases of eclampsia, as also in 
such diseases as diabetes mellitus, chronic alcoholism, phthisis, obesity, 
leukemia, in certain mental diseases, in affections of the pancreas 
and heart, etc. 

The term chyluria or galacturia has been applied to a condition 
in which a turbid urine presenting the macroscopic appearance of 
milk is excreted. Upon microscopic examination it may be demon- 
strated that the turbidity in such cases is owing to the presence 



1 Trans. Assoc. Amer. Phys., vol. xv. 

2 Amer. Jour., December, 1904, p. 1038. 

3 ■ 



Arch. f. exp. Pathol, and Pharmak., 1899, vol. xlii, p. 149, and ibid., 1900- 
1901, vol. xlv, p. 388. 



540 THE URINE 

of innumerable highly refractive globules of fat, which may be 
removed by shaking with ether. Of morphological constituents, 
leukocytes are occasionally encountered in large numbers. Red 
blood corpuscles are also seen at times, and when present in large 
numbers impart a rose color to the urine. Fibrinous coagula are 
often observed when the urine has stood for some time, and the 
entire bulk of urine may even become transformed into a gelatinous 
mass. Albumin is present in most cases in the absence of other 
constituents pointing to renal disease, such as tube casts and renal 
epithelial cells. Leucin, tyrosin, and cholesterin may also at times 
be found, particularly the latter. It has been quite generally accepted 
that chyluria is due to the presence of the Filaria sanguinis hominis; 
but while fllarias are undoubtedly present in the blood in the majority 
of instances, and may also be present in the urine, it has been demon- 
strated that cases occur in which filariasis does not exist. 

Literature. — Lipuria: Schiitz, Prag. med. Woch., 1882, vol. vii, p. 322. 
Ebstein, Arch. f. klin. Med., 1879, p. 115. Chyluria: Huber, Virchow's Archiv, 
1886, vol. cvi, p. 126. Rossbach-Gotze, Verhandl. d. Congr. f. inn. Med., 1887, 
vol. vi, p. 212. Brieger, Zeit. f. physiol. Chem., 1880, vol. iv, p. 407. Grim, 
Langenbeck's Archiv, 1885, vol. xxxii, p. 511. 



Ferments. 

Ferments may be demonstrated in every urine, both under physio- 
logical and pathological conditions. Pepsin is said to be absent in 
cases of typhoid fever, carcinoma of the stomach, and possibly also 
in nephritis. In order to demonstrate its presence, a small flake of 
boiled fibrin is placed in the urine, and after several hours removed 
to a 2 to 3 pro mille solution of hydrochloric acid. The pepsin, if 
present, will be deposited upon the fibrin and effect digestion of the 
latter in the hydrochloric acid solution. 

Diastase, a milk-curdling ferment, and a fat-splitting ferment have 
also been observed. It is noteworthy that the fat-splitting ferment 
was first encountered in a case of hemorrhagic pancreatitis, and it 
has been suggested that its presence may possibly be of value in the 
diagnosis of the disease. Opie, who reports the case, demonstrated 
its presence by the method of Kastle and Loevenhart. Only a small 
amount of urine was obtained. This was neutralized with ~ alkali 
and divided into two portions. To one portion were added 0.25 c.c. 
of ethyl butyrate together with a small quantity of litmus solution 
and 0.1 c.c. of toluol. The second portion used as a control was 
boiled in order to destroy the ferment if present, and ethyl butyrate 
added. Both specimens were kept at 37° C; at the end of twenty- 
four hours the unboiled specimen had acquired a well-marked acid 
reaction, while the control specimen was little if at all changed. A 
quantitative estimation can be made by titrating the two specimens 



CHEMISTRY OF THE URINE 541 

with T \ alkali (using phenolphthalein as an indicator), and noting 
the amount of ethyl butyrate which is split by the ferment. The 
titration should be made after adding to each specimen 0.5 c.c. more 
of ^- HC1 than of the T \ alkali originally used, and to shake out the 
butyric acid with 50 c.c. of ether and 25 c.c. of alcohol ; the acid is then 
titrated directly in the ethereal solution. 

Since the diagnosis of acute lesions of the pancreas is difficult and 
at times impossible the demonstration of the constant occurrence of 
the ferment under such circumstances would be of great importance. 
Its diagnostic importance has been further emphasized by the experi- 
mental work of Hewlett on dogs (which see). 

Literature. — Opie, Johns Hopkins Hospital Bull., 1902, vol. xiii, p. 117. 
Kastle and Loevenhart, Amer. Chem. Jour., vol. xxiv. Hewlett, Jour, of Med. 
Research, May, 1904, p. 377. Gamier, Compt.-rend., 1903, vol. v, p. 1064. 



Gases. 

Every urine contains a small amount of gases, notably carbon 
dioxide, oxygen, and nitrogen, which may be withdrawn by means of 
of an air-pump. 

Under pathological conditions hydrogen sulphide is at times also 
found, constituting the condition known as hydrothionuria. In 
some instances this is referable to a diffusion of the gas into the 
bladder from neighboring organs or accumulations of pus; but this 
is rare. In others an abscess has ruptured into the bladder, or a direct 
communication exists between it and the bowel. Under such con- 
ditions it can, of course, not be surprising that hydrogen sulphide 
together with other products of albuminous putrefaction are elimi- 
nated in the urine. More commonly, however, the hydrothionuria 
occurs idiopathically, and is then referable to the action of certain 
microorganisms. This can be readily demonstrated by adding a 
few cubic centimeters of such urine to normal urine, when upon 
standing the formation of hydrogen sulphide may be demonstrated 
in the normal specimen. The common organisms, however, which 
cause ammoniacal decomposition apparently have no part in this 
process, and the formation of the hydrogen sulphide may be ob- 
served before ammoniacal decomposition has set in, and while the 
reaction is yet acid. If a small amount of ordinary decomposing 
urine, moreover, is added to fresh normal urine, no hydrogen sul- 
phide is, as a rule, produced. The character of the organisms in 
question is variable; sometimes micrococci are found, at other times 
bacilli, and in still other instances both. Besides being capable of 
producing hydrogen sulphide from the sulphur bodies of the urine, 
some of them also cause the formation of ammonium carbonate in 
dilute solutions of urea. 



542 THE URINE 

The source of the hydrogen sulphide in cases of hydrothionuria 
is in most cases probably the so-called neutral sulphur, but it is pos- 
sible that the oxidized sulphur is at times also attacked. In cystinuria, 
in which the neutral sulphur is more or less increased, hydrothionuria 
is commonly observed. Its occurrence in such cases is indeed so 
frequent that I am inclined to suspect cystinuria, although crystals 
of cystin are not found in the sediment. 

In a few recorded instances the hydrothionuria accompanied indigo- 
suria, viz., the presence of free indigo blue in the urine; and this 
Miiller has likewise shown to be referable to the action of certain 
microorganisms. (See Indigosuria.) 

The formation of hydrogen sulphide in decomposing urines con- 
taining albumin is, of course, common, and should not be confused 
with the idiopathic hydrothionuria here described. 

The chemical test for hydrogen sulphide is very simple: a strip 
of filter paper is moistened with a few drops of sodium hydrate and 
lead acetate solution and clamped into the neck of the bottle contain- 
ing the urine. After a variable length of time, in some instances 
immediately, in others only after twelve to twenty-four hours, a dis- 
coloration of the paper will be observed, varying from a grayish 
brown to black according to the amount present. When this is large 
it is, of course, also recognized by its characteristic odor. 

Literature. — F. Miiller, "Schwefelwasserstoff im Harn," Berlin, klin. Woch., 
1887, Nos. 23 and 24. Rosenheim u. Gutzmann, Deutsch. med. Woch., 1888, 
No. 10. Kahler, Prag. med. Woch., 1888, No. 50. 

Ptomains. 

Numerous researches have shown that traces of toxic alkaloidal 
substances may be encountered in the urine under the most diverse 
pathological conditions, and may be present even in health. Of 
the nature of these bodies, however, little is known. Thudichum 
claims to have isolated three distinct basic substances from normal 
urine, which he has termed reducin, parareducin, and arom'in. 
Pouchet and Mme. Eliacbeff, working in Gautier's laboratory, have 
likewise extracted toxic bodies from normal urines; and Adduco 
states that after fatiguing exercise, especially, he could demonstrate 
in the urine a substance which was extremely toxic, and was not 
identical with' cholin, as was first supposed. All this work, however, 
must be repeated with great care before the results obtained can be 
regarded as conclusive. This is also true of the work which has 
been done in various diseases. Some observers have here described 
bodies which they regard as specific toxins. Griffith thus reports 
the presence of a specific poison of scarlatina, of measles, mumps, 
etc. His results, however, do not invite confidence and have never 
been confirmed either by himself or by others. 



CHEMISTRY OF THE URINE 543 

The only substances belonging to the class of ptomains which have 
thus far been obtained from the urine in amounts sufficient to estab- 
lish their identity are cadaverin and putrescin. They were originally 
discovered by Brieger in putrefying cadavers, and subsequently also 
found in cultures of the bacillus of Asiatic chdlera, the Finkler- 
Prior bacillus of cholerina, the bacillus of tetanus, and in the rice- 
water stools of cholera patients. From the urine cadaverin, putrescin, 
and a third diamin isomeric with cadaverin, which has been regarded 
as saprin or neuridin, were first obtained by Baumann and v. Udrans- 
zky in a case of cystinuria, and it appears that diaminuria occurs 
only in association with this disease. All attempts to isolate diamins 
from the urine under other pathological conditions at least have 
given rise to negative results. Regarding the origin of the ptomains 
in question there can be no doubt, I think, that they are derived 
from the corresponding hexon bases, arginin and lysin, as the result 
of a definite metabolic anomaly, of which the cystinuria is also one 
expression. I have advocated this view for some years, and Lowy 
and Neuberg have recently furnished the experimental proof for 
this supposition. They found in a cystinuric individual who w T as not 
excreting any diamins that putrescin and cadaverin appeared when 
the corresponding hexon bases were ingested. Lowy and Neuberg 
further claim to have found tyrosin and aspartic acid when these 
were given by the mouth, which would tend to show that in the cysti- 
nuric there is even a more extensive inability to oxidize amino-acids 
than the cystinuria and diaminuria alone would indicate. I have not 
been able to verify these findings, however, so far as tyrosin is con- 
cerned, and Folin also obtained negative results. 

Putrescin has been found by Baumann and v. Udranszky, Bodtker, 
and Garrod. Brieger, Stadthagen, Leo, Garrod, Lewis, and I have 
succeeded in isolating cadaverin from such urines. Others have been 
less successful. As regards the question whether diaminuria and 
cystinuria invariably coexist I have shown that this is not always 
so, and that the two conditions may alternate, and that the one may 
temporarily disappear while the other continues. Whether or not 
cases occur in which diamins are constantly absent I am not pre- 
pared to say. Cases have been reported by Garrod and others in 
which no diamins could be found, but it is possible that our analy- 
tical methods are not sufficiently delicate to demonstrate mere traces. 
The amount of diamins which may be met with in the urine of 
cystinuric patients is extremely variable. In one case I was able 
to isolate 1.6 grams of the benzoylated cadaverin from the collected 
urine of twenty-four hours. On other days traces only were present, 
and at times no diamins at all could be found. In the case of Dr. 
Lewis, I obtained only 0.3 gram from 12,000 c.c. 

Isolation of Diamins. Method of Baumann and v. Udranszky. — 
The collected urine of at least twenty-four hours is shaken with a 



544 THE URINE 

10 per cent, solution of sodium hydrate and benzoyl chloride in the 
proportion of 1500 to 200 to 25 until the odor of the benzoyl chloride 
has entirely disappeared. The resulting precipitate contains phos- 
phates, the benzoyl compounds of the normal carbohydrates of the 
urine, and a portion of the benzoylated diamins. These are filtered 
off with the aid of a suction pump and digested with alcohol. The 
filtered alcoholic extract is concentrated to a small volume and 
poured into about 30 times its amount of water. Upon standing for 
from twelve to forty-eight hours the benzoylated diamins separate out 
in the milky fluid in the form of a more or less voluminous sediment 
composed of fine, intensely white crystals. In order to remove 
the benzoylated carbohydrates likewise present, the precipitate is 
redissolved in alcohol, the solution concentrated to a small volume, 
and diluted with water as described. This process is repeated several 
times. The resulting crystals, if both diamins are present, will lose 
their water of crystallization at 120° C. and melt at 140° C. 

A smaller portion of the benzoylated diamins remains in the first 
filtrate. In order to recover this the filtrate is acidified with sulphuric 
acid and extracted with ether. The ethereal residue, before congeal- 
ing, is placed in as much of a 12 per cent, solution of sodium hydrate 
as is required for its neutralization, when from 3 to 4 times the 
volume of the same solution is added. This mixture is placed in 
the cold, when long needles and platelets separate out, which consist 
of the sodium compound of benzoyl cystin and the benzoylated dia- 
mins. The sediment is filtered off and placed in cold water, in which 
the sodium-benzoyl cystin dissolves, while the benzoylated diamins 
remain undissolved. 

In order to separate the putrescin from the cadaverin, the crystals 
are dissolved in a little warm alcohol and treated with 20 times the 
volume of ether. Benzoyl putrescin is thus thrown down, and may 
be recognized by its melting point, viz., 175° to 176° C, while the 
ethereal residue contains the benzoyl cadaverin, which melts at from 
129° to 130° C. 

The diamins may then be separated from the benzoyl radicle by 
heating the crystals on a water bath with a mixture of equal parts 
of alcohol and concentrated hydrochloric acid until a specimen is 
entirely dissolved by sodium hydrate. The separation is complete 
after from twenty-four to forty-eight hours, according to the amount 
present. The solution is then diluted with water, when the benzoic 
acid, which has been formed, separates out and is filtered off. After 
extracting with ether, in order to remove any benzoic acid still remain- 
ing, the filtrate is evaporated to dryness. A crystalline mass remains, 
which is easily soluble in water, but with difficulty in alcohol. This 
consists of putrescin and cadaverin hydrochlorates, from which the 
various double salts with platinum, silver, mercury, etc., can be 
readily obtained. The platinum salt of cadaverin is formed by add- 



CHEMISTRY OF THE URINE 545 

ing an alcoholic solution of platinum chloride to a solution of the 
hydrochlorate in alcohol; it occurs as a voluminous yellow, crystalline 
mass, which can be purified by recrystallization from hot water. 
When this salt is decomposed by hydrogen sulphide the hydrochlorate 
again results, from which the free base is obtained by distillation with 
caustic potash. During this distillation water passes over at first; 
and above 160° C. a colorless oil appears, the boiling point of which 
is about 173° C. This constitutes the free base, which may be 
identified by its sperm-like odor and the avidity with which it attracts 
carbon dioxide from the air to form carbonate. 

Phenylcyanate Method (Lowy and Neuberg). — This method has 
certain advantages over the one preceding and may be utilized in 
doubtful cases. In aqueous solution phenylcyanate does not unite 
with carbohydrates and the cystin derivative does not separate out 
in the presence of free alkali, but only upon the addition of an acid. 

The urine is feebly acidified with sulphuric acid and precipitated 
with phosphotungstic acid. The precipitate is collected on a filter, 
washed with 5 per cent, sulphuric acid, suspended in water, and the 
adherent sulphuric acid counteracted with a little baryta. The solu- 
tion is then heated to 5.0° C. and treated with a concentrated solution 
of barium hydrate, also heated to 50° C. until a slight excess is demon- 
strable in the filtrate by means of sodium carbonate. The precipi- 
tate is removed by filtration and the excess of barium by means of a 
current of carbon dioxide. The resultant solution is treated with nor- 
mal alkali (about 40 c.c), and phenylisocyanate now added drop by 
drop. During this process there is a distinct evolution of heat. A 
voluminous precipitate is formed, which is filtered off. This is almost 
insoluble even in boiling alcohol, but dissolves in pyridin. From the 
resultant solution the phenylcyanate of the diamins separates out upon 
the careful addition of water, in snowy white crystals. They can be 
purified by repeated solution and reprecipitation. The putrescin 
compound can be separated from the cadaverin derivative by adding 
water-free acetone to the concentrated solution in pyridin, when the 
putrescin phenylcyanate is thrown down at once, while the other only 
separates from the filtrate after standing for several hours. The 
melting point of the cadaverin compound is 207° C. and of the 
putrescin derivative 238° to 240° C. 

Literature. — Stadthagen, "Ueber d. Harngift," Zeit. f. klin. Med., 1889, vol. 
xv, p. 383. Bouchard, Compt.-rend. Soc. de biol., 1884; and Compt.-rend. de 
l'Acad. des sci., vol. cii, p. 1127. Lepine et Aubert, ibid., vol. ci, p. 90. Adduco, 
Arch, ital. d. Biol., vol. ix, p. 203, and x, p. 1. 

Diaminuria: v. Udranszky u. Baumann, Zeit. f. physiol. Chem., 1889, vol. xiii, 
p. 562. Stadthagen u. Brieger, Berlin, klin. Woch., 1889, vol. xxvi, p. 344. 
Bodtker, Norsk. Mag. f. Laegevidensk., 1892, vol. vii, p. 1220. Moreigne, Arch, 
de Med. exper. et d'Anat. path., 1899, p. 254. Simon, Amer. Jour. Med. Sci., 1900, 
vol. cxix, p. 39. Garrod and Cammidge, Jour. Path, and Bact., Feb., 1900. 
Bodtker, Zeit. f. phys. Chem;, 1905, vol. xlv, p. 393. Lowy and Neuberg, Zeit. 
f. phys. Chem., 1904, vol. xliii, p. 338. 
35 



546 THE URINE 



KRYOSCOPIC EXAMINATION OF THE URINE. 

The kiyoscopic examination of the mixed urine does not furnish 
as valuable information as the corresponding examination of the 
blood. This is largely owing to the fact that the normal variations 
in the freezing point of the urine are much more extensive — i. e., 
between — 0.9° and — 2° C. In the determination of renal insuffi- 
ciency, however, where specimens from each kidney separately are 
available, or at least one specimen from one kidney together with 
a mixed specimen from the same patient, the method furnishes 
very satisfactory results; it indicates the location of the disease 
more definitely than a quantitative estimation of urea, tests of specific 
gravity, and the other usual tests of the urine. Especially interest- 
ing are the results which are obtained in cases of unilateral disease 
of the kidneys in which the other organ is functioning normally; 
kryoscopic examination of the blood will then furnish normal values 
as there is normal elimination, while a separate examination of the 
urine from the two sides reveals the diseased kidney. A value 
of J higher than — 0.9° C. is abnormal. 

In pneumonia, Schmidt found the freezing point much lowered. 
It does not rise to normal until after that of the blood, i. e., several 
days after the crisis. 

The examination is conducted as described in the case of the 
blood. 

Literature. — See Kryoscopy of the Blood. 



MICROSCOPIC EXAMINATION OF THE URINE. 

Sediments. 

In the chapter treating of the general physical characteristics of 
the urine it was stated that, on standing, every urine gradually be- 
comes cloudy owing to the development of the so-called nubecula. 
This was shown to consist of a few mucous corpuscles, a small number 
of pavement epithelial cells derived from the urinary and genital 
passages, and under certain conditions of a few crystals of uric acid, 
of calcium oxalate, or of both. It was further pointed out that 
owing to a diminution in the acidity of the urine on standing, in 
consequence of an interaction between the neutral sodium urate and 
the acid sodium phosphate, a sediment is thrown down which con- 
sists of acid sodium urate, and at times of free uric acid (see Reac- 
tion). Should the reaction of the urine be alkaline, however, when 
freshly voided, a condition which may occur physiologically, when 



MICROSCOPIC EXAMINATION OF THE URINE 547 

it is dependent upon the ingestion of large quantities of vegetables 
rich in organic salts of the alkalies, but which may also be due to 
ammoniacal decomposition, those constituents of the urine which are 
held in solution merely in consequence of the presence of acid sodium 
phosphate are also thrown down. In that case the sediment consists 
essentially of calcium, magnesium, and ammonium salts. Crystals of 
ammonio-magnesium phosphate, it is true, may also be observed in 
alkaline urines of the first variety, but they are then almost always 
due to an increased elimination of ammonia, and hence are rarely 
observed under physiological conditions. 

Normally calcium is found only in combination with phosphoric 
acid and carbonic acid. Of the three possible calcium salts of phos- 
phoric acid — i. e., Ca 3 (P0 4 ) 2 , CaHP0 4 , and Ca(H 2 P0 4 ) 2 — only the 
first two are found in an alkaline urine, but they may also be observed 
in specimens which are either neutral or but faintly acid. The acid 
calcium phosphate, Ca(H 2 P0 4 ) 2 , is seen but rarely in sediments; it 
is precipitated together with uric acid and under similar conditions. 
Calcium carbonate, CaCO s , is seen only in neutral or alkaline urines. 
As soon as ammoniacal fermentation has begun, ammonium salts 
are formed, viz., ammonium urate and ammonio-magnesium phos- 
phate. 

The following table shows the various mineral constituents usually 
observed in sediments, the reaction of the urine being in every case 
the all-important factor: 
Reaction acid: 

Uric acid. 

Sodium urate. 

Calcium oxalate. 

Primary calcium phosphate. 

Ammonio-magnesium phosphate. 
Reaction alkaline (referable to fixed alkalies): 

Secondary calcium phosphate. 

Tricalcium phosphate. 

Calcium carbonate. 

Ammonio-magnesium phosphate. 
Reaction alkaline (referable to ammonia) : 

Ammonium urate. 

Ammonio-magnesium phosphate. 

Tricalcium phosphate. 

Calcium carbonate. 
In pathological conditions still other unorganized substances may 
be observed, viz., cystin, xanthin, hippuric acid, indigo, urorubin, 
hematoidin, magnesium phosphate, calcium sulphate, cholesterin, 
leucin, tyrosin, fats, soaps of magnesium and calcium, etc. Of these, 
cystin, xanthin, hippuric acid, tyrosin, calcium sulphate, hematoidin, 
magnesium phosphate, leucin, and the soaps of magnesium and 



548 • THE URINE 

calcium occur principally in acid urines, while indigo, urorubin, and 
cholesterin are usually only found in alkaline specimens. Before 
considering these various constituents in detail, a few words regarding 
sediments in general and the method to be followed in their micro-' 
scopic examination may not be out of place. 

An idea of the nature of a deposit may often be formed by simple 
inspection, especially if the reaction of the urine is known. 

A crystalline sediment, presenting a brick-red color and appearing 
to the naked eye like cayenne pepper, is referable to uric acid. On 
the other hand, a salmon-red, amorphous deposit occurring in an acid 
urine consists essentially of sodium urate. Should doubt be felt, it 
will only be necessary to heat the urine, when the urate deposit will 
dissolve. A white, flocculent sediment in an alkaline urine is usually 
referable to a mixture of phosphates and carbonates, and will dissolve 
upon the addition of acetic acid, but remains unaffected by heat. 

A sediment consisting of pus, and occurring in alkaline urines, is 
frequently mistaken for a phosphatic deposit by the beginner. Aside 
from a microscopic examination, the question may be settled by 
the addition of a small piece of caustic soda and stirring, when in 
the presence of pus the liquid becomes mucilaginous and ropy. If 
much pus is present, a tough, jelly-like mass will be formed, which 
escapes from the vessel en masse when the urine is poured out. 
Such a sediment, moreover, does not disappear upon the addition 
of an acid, and is rendered still more dense upon the application of 
heat. 

Blood when present beyond traces may also be recognized. 

As a general rule, the non-organized elements of a sediment are 
of little clinical interest. 

Students are frequently in the habit of diagnosticating an ex- 
cessive, normal, or subnormal elimination of one or another urinary 
constituent from the result of a microscopic examination. This is 
unwarrantable. It should always be remembered that no con- 
clusions whatsoever can be drawn in this manner as to the amount 
actually eliminated. Nothing would be more erroneous than to infer 
an excessive excretion, not to speak of an excessive production of 
uric acid or of oxalic acid from the fact that crystals of these sub- 
stances are seen in large numbers under the microscope. Again 
and again cases are observed in which an excessive elimination of 
uric acid, oxalic acid, or phosphates is thus diagnosticated in which 
chemical analysis shows not only no increase but even a diminution 
of the normal quantity. 

A urine which is turbid when passed may be examined micro- 
scopically at once. As a rule, however, it is necessary to wait until 
a sediment has formed. To this end the urine should be kept in 
a clean and well-stoppered bottle. A small amount of chloroform 
is added if necessary, and will preserve the specimen almost in- 



MICROSCOPIC EXAMINATION OF THE URINE 



549 



definitely. A few drops of the sediment are then removed by means 
of a clean pipette, carried down to the sediment, with the distal end 
tightly closed by the finger, care being taken not to allow the urine 
to rush into the tube by suddenly releasing the pressure, but with- 
drawing an amount just sufficient for an examination. This is then 
spread over a clean slide that has been moistened by the breath, 
when the specimen may be examined at once. Covering the speci- 
men with a slip is unnecessary. A low power of the microscope (B. 
& L. f ; Leitz 3) should always be employed, and the high povjer 
only used to study details of structure. 








Fig. 151. — Various forms of uric acid crystals. (Finlayson.) 

If a centrifugal machine is available, it is, of course, not necessary 
to let the urine stand until a sediment has formed. An amount 
sufficient for a microscopic examination can then be obtained in a 
few minutes. 



Non-organized Sediments. 

Sediments Occurring in Acid Urines. Uric Acid.— The form which 
uric acid crystals may present in a deposit varies greatly, the most 
common being the so-called whetstone form (Figs. 143 and 151). The 
crystals may occur singly or in groups. Accidental impurities, such as 
threads or hairs, are at times covered with such crystals, forming long 
cylinders. Very frequently uric acid crystallizes in the form of large 
rosettes of drawn-out whetstone crystals, presenting a brownish-red 
color, referable to uroerythrin, when they are often visible to the 
naked eye, and form the well-known brick-dust sediment. While it 
is generally stated that uric acid crystals can always be recognized 
by their color, which may vary from a light yellow to a dark brown, 



550 THE URINE 

this is, in my experience, not the case. I have often seen uric acid 
sediments in which the crystals formed small rhombic plates with 
rounded edges, and were absolutely devoid of coloring matter, so 
far as a microscopic examination could show. Uric acid "dumb- 
bells" are also at times observed, and may be mistaken for calcium 
oxalate. Hexagonal plates of uric acid have been similarly con- 
founded with cystin. 

A uric acid sediment may be observed in cases in which an in- 
creased excretion of uric acid occurs, but it does not follow that a 
uric acid sediment indicates an increased elimination. Such an 
assumption would only be warrantable if a quantitative estimation 
had been made. It is more common to meet with uric acid sedi- 
ments where the actual amount is not increased than the contrary. 
Brick-dust sediments are frequently observed during cold weather, 
but it would be erroneous to infer an increased elimination from such 
an occurrence, as the phenomenon is owing to the fact that uric acid 
is less soluble in cold than in warm water. During the summer 
months, for the same reason, a deposit of uric acid is less frequently 
observed, although an increased amount may nevertheless be present, 
being held in solution owing to the higher temperature. The more 
concentrated the urine, the more readily will such a deposit form. 
It is hence noted after profuse perspiration, following severe muscular 
exercise, in acute rheumatism with copious diaphoresis, in acute 
gastritis and enteritis associated with copious vomiting or diarrhea, 
during the crisis of pneumonia (particularly if accompanied by much 
sweating), etc. 

Where a distinct tendency exists for the separation of uric acid 
sediments, as in cases where the urine is habitually over-acid, the 
possibility of the same occurrence within the urinary passages should 
be borne in mind (gravel, calculus). F. Miiller has recently shown 
that the habitual separation of uric acid from the urine which is noted 
in such cases and which is commonly associated with vague dyspeptic 
and nervous disturbances is referable to the presence in such urines 
of considerable amounts of organic acids of unknown composition. 

Chemically, the nature of a uric acid sediment may be recognized 
by the fact that the crystals dissolve upon the addition of sodium 
hydrate and reappear in the rhombic form upon acidifying with 
hydrochloric acid When heated with dilute nitric acid the beauti- 
ful red color of ammonium purpurate is obtained upon the subsequent 
addition of ammonia (murexid test), as described elsewhere. 

Amorphous Urates, — Sodium and potassium urates frequently, and 
especially in fevers, form sediments of such density that upon micro- 
scopic examination it is almost impossible to discern anything but 
innumerable amorphous granules scattered over the entire field 
and obscuring all other elements that may be present. Cells or 
casts will frequently be seen studded with these granules. In such 



MICROSCOPIC EXAMINATION OF THE URINE 551 

cases it is best to heat the urine to a temperature of 50° C, and to 
centrifugalize the urine as soon as it has cleared. 

Urate sediments are always colored, the tint varying from a dirty 
yellowish brown to a bright salmon red, owing to the presence of 
uroerythrin. Difficulties can hence never arise in determining the 
nature of the sediment, as a colored deposit appearing in an acid 
urine which dissolves upon the application of heat cannot be due to 
anything but urates. If a drop of the sediment, moreover, is treated 
upon a slide with a drop of hydrochloric acid, characteristic whet- 
stone crystals of uric acid separate out, but the greater portion appears 
in the form of rhombic platelets. 

Calcium Oxalate. — This substance generally appears in urinary 
sediments in the form of colorless, highly refractive octahedra (Fig. 
152), which vary greatly in size; some appear as mere specks under 
even a comparatively high power, while others may attain the dimen- 
sions of a large leukocyte. Frequently one axis is longer than the 






Fig. 152. — Calcium oxalate crystals. (Finlayson.) 

other. From the fact that their diagonal planes are highly refractive, 
apparently dividing the superficial plane into four triangles, they 
have been compared to envelopes, and it is this envelope form of the 
crystals which is especially characteristic. In the same specimen of 
urine so-called dumb-bell forms may be seen, which appear to be 
made up of two bundles of needle-like crystals united in the form of 
the figure 8. Other forms may also be seen, and are shown in the 
accompanying figure. 

While the envelope crystals are highly characteristic and can 
hardly be mistaken for any other substance, the student may at times 
confound them with crystals of ammonio-magnesium phosphate. 
This error may be avoided if it is remembered that the calcium oxa- 
late crystals are usually not so large as those of the magnesium salt, 
and that the latter dissolve upon the addition of acetic acid, in which 
calcium oxalate is insoluble. The distinction from uric acid, if we 
are dealing with the dumb-bell form, cannot always be made by 
mere inspection. A drop of caustic soda should be added, which 



552 THE URINE 

will dissolve the crystals if they are uric acid, while calcium oxalate 
remains unchanged. 

It has been pointed out that under strictly normal conditions a 
few isolated crystals of calcium oxalate may be found in the primi- 
tive nubecula, so that their presence in urinary sediments cannot be 
regarded as pathological. After the ingestion of certain vegetables 
and fruits, notably tomatoes, rhubarb, garlic, asparagus, and oranges, 
or following the continued administration of sodium bicarbonate or 
the salts of vegetable acids, calcium oxalate crystals may be observed 
in large numbers; so also in certain diseases, such as diabetes mellitus, 
catarrhal jaundice, phthisis, emphysema, etc. 

As in the case of uric acid, no inference as to the quantity elimi- 
nated can be drawn from a microscopic examination of the sedi- 
ment. The frequent occurrence of abundant sediments of this sub- 
stance may, however, generally be regarded as abnormal, providing 

that such an occurrence cannot be ex- 
plained by the nature of the diet. It is 
very suggestive to note the frequency 
with which such sediments are observed 
in cases of neurasthenia associated with 
a mild degree of albuminuria, as also in 
various digestive neuroses. Finally, as 
with uric acid, the possibility of the 
formation of renal calculi should be 
borne in mind whenever abundant sedi- 
ments of calcium oxalate are encoun- 
tered upon frequent examination. 
riG - 153 pirafe ? r °yTtX mph0S " Monocalcium phosphate crystals are 

rarely seen, and only in specimens pre- 
senting a highly acid reaction, when uric acid crystals are also fre- 
quently observed in large numbers (Fig. 153). They are colorless 
and soluble in acetic acid. 

Hippuric acid crystals have been observed, although rarely, in uri- 
nary sediments, in acute febrile diseases, diabetes, and chorea, while 
their occurrence following the ingestion of large amounts of prunes, 
mulberries, blueberries, or the administration of benzoic acid and 
salicylic acid, is more common. 

Hippuric acid occurs in the form of fine needles or rhombic prisms 
and columns, the ends of which terminate in two or four planes, at 
times resembling the crystals of ammonio-magnesium phosphate and 
of uric acid. From the former they may be readily distinguished by 
their insolubility in hydrochloric acid, and from the latter by the 
fact that they do not give the murexid reaction when treated with 
nitric acid and ammonia. In the case of urines rich in hippuric 
acid in which the substance does not appear in the sediment, it is 
well to add a small amount of hydrochloric acid, when the crystals 




MICROSCOPIC EXAMINATION OF THE URINE 



553 



will gradually separate out. Their presence does not appear to possess 
any clinical significance. 

Calcium sulphate, in the form of long, colorless needles or elon- 
gated prismatic tablets (Fig. 154), has been observed in urinary 
sediments in only two cases. In both the urine, especially on stand- 
ing, deposited a milky-looking sediment, the reaction being strongly 
acid. It may be recognized by its insolubility in acids and ammonia. 1 

Cystin is rarely seen in urinary sediments. It occurs in the form 
of colorless, hexagonal platelets, which are very characteristic (Fig. 
155). The crystals are soluble in ammonia and hydrochloric acid, 
and insoluble in acetic acid, water, alcohol, and ether. They can 
thus be readily distinguished from certain forms of uric acid, with 




Fig. 154.— Calcium sulphate crystals, 
(v. Jaksch.) 



Fig. 155. — Crystals of cystin spontaneously 
voided with urine. (Roberts.) 



which they might possibly be confounded at first sight. When 
heated upon platinum foil they burn with a bluish-green flame with- 
out melting. 

Cystin-containing urines may be of normal appearance, but they 
often present a peculiar greenish-yellow color. The reaction is 
mostly neutral or alkaline. Upon standing exposed to the air a 
marked odor of hydrogen sulphide develops, owing to decomposition 
of the cystin ; but at times urines are met with in which a distinct odor 
of hydrogen sulphide is noticeable, although crystals of cystin are not 
seen in the sediment. It may then be demonstrated by strongly 
acidifying the urine with acetic acid or by allowing it to undergo 
ammoniacal decompostion. In either case cystin crystals will sepa- 
rate out on standing. It should be remembered, however, that not all 
urines in which hydrogen sulphide is formed contain cystin. (See 
Hydro thionuria. ) 

1 v. Jaksch, Zeit. f. klin. Med., 1892, vol. xxii, p. 554. 



554 THE URINE 

The amount of cystin which may be found in urinary sediments 
is variable. Sometimes a few crystals only are obtained, while at 
others from 0.5 to 1 gram may be recovered. As is the case with 
the other non-organized constituents of sediments, however, the 
amount deposited does not necessarily indicate the total amount 
present. Where a quantitative estimation of cystin is to be made, 
it is best to filter off that which is deposited and to estimate the 
amount of neutral sulphur in the filtered urine. An increase beyond 
the normal may be referred to the cystin remaining in solution. (See 
Neutral Sulphur.) 

Clinical interest in connection with cystinuria centres in the fre- 
quent association of cystin sediments with cystin gravel or calculi; 
but the cystinuria may exist for years without giving rise to clinical 
symptoms. 

Very remarkable is the not uncommon occurrence of cystinuria in 
families. Cases of transient cystinuria likewise occur, and it is 
hence scarcely admissible to speak of a "cured" cystinuria when the 
condition disappears under some supposed treatment. 

Of the origin of the condition little is known. It has been sup- 
posed that the appearance of cystin in the urine is in some manner 
connected with the formation of certain diamins in the intestinal 
canal. I have pointed out, however, that in all probability the for- 
mation of cystin and diamins takes place in the tissues of the body, 
and that the appearance of both is the expression of a definite met- 
abolic anomaly rather than of a specific infection. (See Ptomains 
and Neutral Sulphur.) 

Literature. — C. E. Simon, "Cystinuria and its Relation to Diaminuria," Amer. 
Jour. Med. Sci., 1900, vol. cxix, p. 39. See also the literature under Ptomains. 

Leucin and tyrosin are never found in urinary sediments under 
normal conditions. They are seen especially in acute yellow atrophy 
and in some cases of acute phosphorus poisoning. 

Traces of leucin and tyrosin are said to be constantly present also 
in cases of cirrhosis and carcinoma of the liver, in cholelithiasis, 
catarrhal jaundice, Weil's disease, nephritis, cystitis, gout, bronchitis, 
tuberculosis, typhoid fever, hysteria, erysipelas, glucosuria, etc., but 
in many cases the proof has not been properly furnished that the sub- 
stance under examination was really tyrosin. In connection with 
cystinuria the elimination of tyrosin has also been observed, but in 
two cases which I examined in this direction I obtained negative 
results. 

Isolation of Leucin and Tyrosin. — As leucin is hardly ever found in 
the sediment, and tyrosin only when present in large quantities, the 
urine in every case should first be concentrated upon a water bath 
and examined on cooling. At times, however, when these substances 
are present in only very small quantities, this procedure may not lead 



MICROSCOPIC EXAMINATION OF THE URINE 



555 



to the desired end, and in doubtful cases the following method should 
be employed: 

The total amount of urine voided in twenty-four hours is pre- 
cipitated with basic lead acetate and filtered, when the filtrate, from 
which the excess of lead has been removed by means of hydrogen 
sulphide, is evaporated to as small a volume as possible. Any urea 
that may be present is removed by shaking with a small amount of 




Fig. 156. — Tyrosin crystals. (Charles.) 

absolute alcohol, and the insoluble residue extracted with alcohol 
containing a little ammonia. This extract is concentrated to a small 
volume and left to spontaneous crystallization. Leucin and tyrosin 
separate out and can then be further examined. 

Ulrich advises to evaporate the urine to dryness and to heat the 
residue gently while the vessel is covered with a plate of glass or a 
funnel. The tyrosin is then said to sublime, and is deposited on the 




Fig. 157.; — Crystals of leucin (different forms). (Crystals of kreatinin-zinc chloride resem- 
ble the leucin crystals depicted at a.) The crystals figured to the right consist of compara- 
tively impure leucin. (Charles.) 

cool glass in crystalline form, the crystals giving the characteristic 
reactions. 

Tyrosin crystallizes in the form of very fine needles (Fig. 156), 
which are usually grouped in sheaves. They are insoluble in acetic 
acid, but soluble in ammonia and hydrochloric acid. 

Leucin (Fig. 157) occurs in the form of spherules of variable size, 



556 THE URINE 

which resemble globules of fat, but may be distinguished from these 
by their insolubility in ether. In the urine they present a more or 
less pronounced brownish color, and upon close examination con- 
centric striations as well as very fine radiating lines can at times be 
made out, which are especially characteristic. 

If crystals resembling tyrosin and leucin are found, the following 
tests should be made: 

Separation of the Tyrosin. — The crystals are filtered off, washed 
with water, and dissolved in ammonia to which a little ammonium 
carbonate has been added. The solution is allowed to evaporate, 
when the tyrosin separates out. 

Piria's Test. 1 — A bit of the tyrosin is moistened on a watch-crys- 
tal with a few drops of concentrated sulphuric acid, covered, and 
set aside for half an hour. It is then diluted with water, heated, 
and while hot saturated with calcium carbonate and the solution 
filtered. The filtrate is colorless, but when heated with a few drops 
of a very dilute neutral solution of ferric chloride it assumes a violet 
tint. 

Hoffmann's Test. 2 — A small amount of tyrosin is dissolved in hot 
water and treated, while hot, with mercuric nitrate and potassium 
nitrite. The solution assumes a dark-red color and yields a volumi- 
nous red precipitate. 

Separation of the Leucin. — After filtering off the tyrosin from the 
ammoniacal solution (see Separation of Tyrosin, above) the residual 
fluid is concentrated to the point of crystallization and treated with 
a little alcohol. This will take up the leucin. The alcoholic extract 
is allowed to evaporate and the residue examined with Scherer's 
test. 

Scherer's Test. 3 — When leucin is treated upon platinum foil with 
nitric acid, a colorless residue is obtained which, upon the applica- 
tion of heat and the addition of a few drops of a solution of sodium 
hydrate forms a droplet of an oily fluid which does not adhere to the 
platinum. 

Hofmeister's Test. 4 — A small amount of leucin dissolved in water 
causes a deposit of metallic mercury when heated with mercurous 
nitrate. 

Literature. — Frerichs, Wien. med. Woch., 1854, vol. iv, p. 465. Schultzen 
u. Riess, Charite Annal., vol. xv. Pouchet, Maly's Jahresber., 1880, vol. x, p. 
248. Irsai, ibid., 1885, vol. xiv, p. 451. Prus, ibid., 1888, vol. xvii, p. 345. 
Frankel, Berlin, klin. Woch., 1878, vol. xv, p. 265. 

Xanthin crystals (Fig. 158) are very rarely observed in urinary 
sediments, and, so far as I have been able to ascertain, the case 

1 Liebig's Annal., 1852, vol. lxxxii, p. 251. 

2 Ibid., 1857, vol. lxxxvii, p. 124. 

3 Jour. f. prak. Chem., 1887, vol. Ixxix, p. 410. 

4 Liebig's Annal., 1877, vol. cxxxix, p. 6. ' 



MICROSCOPIC EXAMINATION OF THE URINE 



557 



observed by Bence Jones 1 is the only one on record. Care should 
be had not to confound colorless crystals of uric acid with xanthin. 
Xanthin is readily soluble in ammonia. It can be identified by means 
of Strecker's test (which see). 

Clinically, xanthin sediments are of interest only in so far as this 
substance may give rise to the formation of calculi. 

Soaps of Lime and Magnesia. — v. Jaksch has pointed out that in 
various diseases crystals may be found which "closely" resemble 
tyrosin in appearance, and pictures such crystals (Fig. 159), which 
from their behavior toward reagents he is inclined to regard as cal- 
cium and magnesium salts of certain higher fatty acids. 

Should doubt arise, the question may be readily decided by a 
chemical examination. (See Tests for Tyrosin and Fatty Acids.) 




'^t/U* 



Fig. 158. — a, crystals of xanthin (Salkow- 
ski); 6, crystals of cystin. (Robin.) 




Fig. 159. — Lime and magnesium soaps. 
(v. Jaksch.) 



Bilirubin (Hematoidin) crystals in the form of yellow or ruby-red 
rhombic plates or needles, as well as amorphous granules, have been 
seen in the urine in rare cases. They are easily soluble in alkalies 
and chloroform, but not in ether. When treated upon a slide with 
a drop of nitric acid a green ring will be seen to form around them 
(Gmelin's reaction). 2 Such crystals have been found either free or 
embedded within cells or tube casts, in cases of scarlatinal nephritis, 
the nephritis of pregnancy, in granular atrophy, amyloid degenera- 
tion of the kidneys, in icteric urines and in carcinoma of the bladder, 
of which latter condition they have been regarded by some as pathog- 
nomonic. 

Fat. — When small, strongly refractive globules of fat, which may 
be readily recognized by their solubility in ether, are observed either 



1 Chem. Centralbl., 1868, vol. xiii. 

2 Kussmaul, Wiirzburgermed. Zeit., 1863, vol. iv, p. 64. 



klin. Med., 1879, vol. xiii, p. 115. 



Ebstein, Arch. f. 



558 THE URINE 

floating on the urine or held in suspension, it is necessary to ascer- 
tain first of all whether such fat may not be present accidentally, 
owing to the use of a bottle or vessel not absolutely clean, or pre- 
vious catheterization, etc. The diagnosis lipuria should only be 
made when all possible precautions have been taken to exclude 
the accidental presence of this substance. True lipuria — i. e., an 
elimination of fat usually in the form of droplets floating on the 
urine — has been noted in various cachectic conditions, in cases of 
heart disease, affections of the pancreas and liver, in gangrene and 
pyemia, in diseases of the bones, especially following fractures, in 
diseases of the joints, in diabetes, and notably in chyluria. Fat has 
also been observed in the urine following the ingestion of large 
amounts of cod-liver oil and inunctions with fats and oils. 

In fatty degeneration of the kidneys (nephritis, phosphorus poison- 
ing, etc.), droplets of fat may be seen in the epithelial cells and tube 
casts. This, however, does not constitute lipuria. The nature of 
the droplets may be recognized by their solubility in ether, benzol, 
chloroform, carbon disulphide, xylol, etc., and by the fact that they 
are colored black when treated with a 0.5 to 1 per cent, solution of 
osmic aid, and red when a drop of tincture of alcanna is added to the 
specimen. A convenient method of demonstrating the presence of 
fat is also the following: A few cubic centimeters of the urine are 
mixed with an equal volume of 96 per cent, alcohol and a concentrated 
solution of Sudan III in 96 per cent, alcohol. The sediment which 
collects is then examined under the microscope; the excess of stain is 
removed by allowing a few drops of 60 or 70 per cent, alcohol to run 
under the cover-slip and removing it with filter paper placed at the 
edge of the preparation. The fat droplets are thus colored a ver- 
milion red. Free fat can, of course, be demonstrated in the same 
manner. (See also Lipuria.) 

Sediments Occurring in Alkaline Urines. Basic Phosphate of Cal- 
cium and Magnesium. — The most common sediments observed in 
alkaline urines consist of amorphous phosphates of calcium and 
magnesium. They are usually as abundant as the urate sediments 
which have been described, but may be distinguished from these by 
the fact that they do not dissolve upon the application of heat, but 
disappear upon the addition of acetic acid, and are never colored. 
In this manner it is also easy to distinguish such a sediment from one 
due to pus, with which it might possibly be confounded at first sight. 
Upon microscopic examination a drop of the sediment will be seen 
to contain innumerable transparent granules scattered over the entire 
field, and closely resembling those of urate of sodium. 

Phosphatic sediments are observed, as mentioned elsewhere, when- 
ever the reaction of the urine is alkaline, whether this be owing to the 
presence of fixed alkali or to ammoniacal fermentation. They also 
result if a faintly acid, faintly alkaline, or amphoteric urine is boiled. 



MICROSCOPIC EXAMINATION OF THE URINE 



559 



Neutral Calcium Phosphate. — These crystals may be found in alka- 
line, amphoteric, and feebly acid urines, but are not very common. 




Fig. 160. — Crystalline phosphates. (Finlayson, 

They are at times of large size, but more commonly acicular, occur- 
ring either singly or united in a star-like manner (Fig. 160). They 
are colorless, readily soluble in acetic 
acid, and insoluble in warm water, so 
that they can be easily distinguished 
from uric acid. 

Basic magnesium phosphate crystals 
occurring in the form of large, highly 
refractive plates (Fig. 161), are at times 
seen in alkaline, neutral, or faintly acid 
and highly concentrated urines. They 
are readily recognized by treating a drop 
of the sediment upon a slide with a drop of ammonium carbonate solu- 
tion (1 to 4), when the crystals become opaque and their edges assume 




Fig. 161. — Basic magnesium phos- 
phate crystals, (v. Jaksch.) 




Fig. 162. — Various forms of triple phosphates. (Finlayson.) 

an eroded aspect. In acetic acid they dissolve with ease and may then 
be reprecipitated by means of sodium carbonate. They are uncommon. 



560 THE URINE 

Ammonio-magnesium phosphate, usually spoken of as triple phos- 
phate, crystallizes in large prismatic crystals of the rhombic system; 
it is most abundantly observed in alkaline urines, but may also occur 
in feebly acid specimens. Of the various forms which may occur, 
that resembling the lid of a German coffin is the most characteristic 
(Fig. 162). At times these crystals attain a large size; very small 
specimens, however, also occur which may be mistaken for oxalate 
of calcium, but from these they are distinguished by the ease with 
which they dissolve in acetic acid. 

Here, as elsewhere, it should be remembered that no conclusions 
as to the amount actually eliminated can be drawn from a micro- 
scopic examination and the diagnosis " phosphaturia" should be 
based only upon the results of a quantitative analysis. 

The continued elimination of a turbid urine, the turbidity of which 
is referable to phosphates, is notably observed in certain neurasthenic 





Fig. 163. — Calcium carbonate Fig. 164. — Ammonium urate 

crystals. crystals. 

individuals with a predominance of cerebral symptoms. Very curi- 
ously the phosphaturia is not influenced by diet. 

Calcium carbonate frequently occurs in alkaline urines, and appears 
under the microscope in the form of minute granules, occurring 
singly or arranged in masses; dumb-bell forms are also seen (Fig. 
163). They may be recognized by the fact that they readily dis- 
solve in acetic acid, with the evolution of gas. 

Ammonium urate is observed only in urines which are undergoing 
ammoniacal decomposition. Its presence should always call for a 
careful investigation in order to ascertain whether this has taken 
place after the urine has been voided or before. (See Reaction.) 

The salt occurs in the form of brownish, spherical bodies of variable 
size, which are sometimes composed of delicate needles, while at 
others they are amorphous, but may be beset with prismatic spicules, 
(thornapple forms). They are not easily mistaken for any other 
substance which may be present in urinary sediments (Fig. 164). 



MICROSCOPIC EXAMINATION OF THE URINE 561 

Ammonium urate is characterized, moreover, by its solubility in 
acetic and hydrochloric acids, and by the subsequent separation of 
rhombic crystals of uric acid. 

Indigo in the form of delicate blue needles, arranged in a stellate 
manner or in plates, visible only with the microscope, is rarely seen. 
In an amorphous condition, however, it may be met with in almost 
every decomposed urine, occurring in the form of small granules 
and sometimes staining the morphological elements that may be 
present a distinct blue. Sediments presenting a bluish-black color 
were noted in the time of Hippocrates already, and have been 
described since by numerous observers, but the nature of the color- 
ing matter has only been determined within the last fifty years. 
Clinically, the occurrence of indigo in the urine is of interest, as 
renal calculi have been observed which consisted almost entirely of 
this substance. But little is known of the causes which give rise 
to its appearance in the urine, but there can be no doubt that its 
occurrence is referable to the action of certain microorganisms upon 
urinary indican (which see). 1 



Organized Constituents of Urinary Sediments. 

Epithelial Cells (Fig. 165). — Bearing in mind the fact that desqua- 
mative processes are constantly going on in the epithelial lining of 
the various cavities and channels of the body, one should expect to 
find in every urine representatives of the different forms of epithelium 
from the Malpighian tufts down to the meatus urinarius. To a cer- 
tain extent this actually happens, and cells apparently derived from 
the meatus, the urethra, bladder, ureters, and pelvis of the kidneys 
may be met with in almost every specimen, although it is often diffi- 
cult to tell the origin of the individual cells. Bizzozero even claims 
that it is impossible to distinguish between the cells of the bladder and 
those of the meatus and renal pelvis, while as a class they may be 
differentiated in most cases from the cells of the urethra, the ureters, 
the prepuce of the male, and the vulva and vagina of the felnale. 
Cells from the uriniferous tubules are seldom seen in normal urines. 

The number of epithelial cells occurring in urinary sediments under 
physiological conditions is small, and the presence of large numbers 
may hence always be regarded as abnormal. Their appearance is 
influenced by the reaction of the urine, an alkaline or neutral urine 
causing them to swell and to appear larger and rounder than in acid 
urines. As has been mentioned, the cellular type is practically the 
same, moreover, in the bladder, ureters, and pelvis of the kidneys. 

As has already been stated it may be very difficult to determine 

1 v. Jaksch, Prag. med. Woch., 1892, vol. xvii, p.. 602. 
36 



562 THE URINE 

the origin of single epithelial cells, or even of groups of cells, by 
examining these per se. But not infrequently other findings may 
lead to their proper classification and interpretation. 

Generally speaking three forms of epithelial cells may be found in 
urinary sediments, viz.: 

1. Round cells. 

2. Conical and caudate cells. 

3. Flat cells. 



Fig. 165. — Urinary epithelium. 

Round cells may be derived from the uriniferous tubules or the 
deeper layers of the mucous membrane of the pelvis of the kidneys. 
They are somewhat larger than pus corpuscles and may be dis- 
tinguished from these by the presence of a large, well-defined nucleus, 
which is readily visible as such, while in pus cells it becomes distinct 
only upon the addition of acetic acid, and is, moreover, multiple. 
Whenever such cells are found adhering to urinary casts, it is clear 
that they represent the glandular elements proper of the kidneys. 
As similar cells are found in the male urethra, confusion may arise. 
Should albumin be present, the cells are probably of renal origin. 
The presence of such cells in large numbers together with pus, in the 
absence of tube casts and albumin beyond traces, will usually indi- 
cate the existence of a simple pyelitis, particularly if round cells are 
found joined in a shingle-like manner. Should the pyelitis be asso- 
ciated with a nephritis, tube casts and albumin in larger amounts 
will at the same time be present. In such cases it may be impossible 
to determine the origin of the cells, excepting of such that may 
adhere to casts. 



MICROSCOPIC EXAMINATION OF THE URINE 563 

In simple circulatory disturbances affecting the renal parenchyma 
no special abnormalities can be discovered in the structure of the 
cells, while in fatty degeneration of the kidneys they will be seen to 
contain fatty particles in greater or less abundance. At other times 
they are markedly granular and occur in fragments*. 

Conical and caudate cells are mostly derived from the superficial 
layers of the pelvis of the kidneys, and are hence seen in large num- 
bers in cases of pyelitis. Similar cells, however, are also found in 
the neck of the bladder. 

Flat cells may come from the ureters, the bladder or the genitals. 
Large polygonal cells provided with single distinct nuclei and a more 
or less markedly granular protoplasmic zone about the nucleus are 
usually derived from the external genitals. Many such cells are 
more or less broken down and distorted. The surface cells from the 
bladder and ureters are less apt to show evidence of injury or degenera- 
tion, and are on the whole smaller. Surface epithelial cells from the 
vagina are mostly fusiform in shape and very commonly show an 
irregular, warped outline. Often they are seen in large plaques. 
Other more or less rounded forms are derived from the deeper layers 
of the mucosa. Irregular or conical cells, often provided with one 
or more protoplasmic processes, likewise come from the lower layer 
of the mucosa of the bladder and ureters. 

In alkaline urines undergoing bacterial decomposition it is com- 
mon to meet with large surface epithelial cells from the external 
genitals which are literally one mass of bacteria. 

Literature.— -Bizzozero, loc. cit. Eichhorst, Lehrbuch d. physikal. Unter- 
such. inn. Krankheit., 2d ed., p. 336, Braunschweig. 

Leukocytes. — Leukocytes are encountered in only very small num- 
bers in normal urines. A marked increase should, hence, always 
be regarded as indicating the existence of disease somewhere in the 
urinary tract, excepting in females, where their presence may be 
owing to an admixture of leucorrheal discharge. In that case the 
source of the pus will generally be recognized by the simultaneous 
occurrence of pavement epithelial cells of the vaginal type in cor- 
respondingly large numbers. In doubtful cases the urine should 
always be obtained with the catheter, care being taken to thoroughly 
cleanse the vulva before the introduction of the instrument. 

Occasionally the pus is derived from a neighboring abscess that 
has opened into the urinary passages. 

The amount of pus which may be found in urines is most varia- 
ble. On the one hand, deposits several centimeters in height are not 
uncommon, and closely resemble deposits of phosphates, for which 
they are indeed frequently mistaken; on the other hand, it may only 
be^ possible to discover the presence of pus by means of the micro- 
scope, which should be employed in every case. 



564 THE URINE 

The appearance of the pus corpuscles varies in different cases. 
In acid urines their form is usually well preserved, and in feebly 
alkaline and neutral specimens it may even be possible to observe 
ameboid movements when the slide is carefully warmed. In alka- 
line urines, however, they usually swell up and become opaque, so 
that it is impossible to discern a nucleus unless they are treated with 
acetic acid. At other times, and particularly when pus has remained 
long in the body, it may be almost impossible to make out a nucleus, 
and in extreme instances nothing but a mass of granular and fatty 
detritus is left. 

While with a certain amount of experience it is hardly'likely that 
a sediment of pus will be mistaken for anything else, it should be 
remembered that if pus is exposed to the action of ammonia or an 
ammonium salt the pus corpuscles become disintegrated. In such 
cases, as in old, neglected instances of cystitis, in which ammoniacal 
decomposition of the urine has taken place in the bladder, a deposit 
may be obtained which microscopically resembles mucus, and in 
which pus corpuscles may not even be demonstrable with the micro- 
scope. The sediment escapes as a gelatinous, slippery mass when 
the urine is poured from one vessel into another. Recourse must 
then be had to certain chemical tests . as a pyuria might otherwise be 
overlooked. To this end the following procedure, suggested by 
Vitali, 1 may be employed: 

The urine, after having been acidified with acetic acid, is filtered, 
and the contents of the filter treated with a few drops of tincture 
of guaiacum which has been kept in the dark, when in the pres- 
ence of pus the filter paper is colored a deep blue. 

A solution of iodopotassic iodide may be employed in less extreme 
instances. A drop of this solution is added to a drop of the sediment 
upon a slide, when the pus corpuscles, owing to the presence of glyco- 
gen, are colored a dark mahogany-brown, while epithelial cells, with 
certain forms of which they might possibly be mistaken, assume a 
light-yellow color. 

Donne's pus test is based upon the fact that the transformation of 
pus ino a gelatinous, mucus-like mass, observed in cases of cystitis, 
owing to the action of ammonium carbonate, may also be artificially 
produced by the addition of a small piece of caustic soda and stir- 
ring, when in the presence of pus in small amounts the liquid becomes 
mucilaginous and ropy, while a gelatinous mass is obtained if it is 
abundant. 

Midlers Modification of Donne's Test. — 5 to 10 c.c. of urine are 
treated drop by drop with official sodium hydrate solution, shaking 
thoroughly after the addition of each drop. If then the tube is 
observed, it will be noted that the bubbles of air can rise only very 

1 Maly's Jahresber., 1890, vol. xviii, p. 326. 



MICROSCOPIC EXAMINATION OF THE URINE 565 

slowly through the viscid fluid or in the presence of fair amounts of 
pus may remain stationary altogether. A positive reaction is still 
obtained from 1200 pus cells to the cb. mm. 

From a clinical point of view it is important to establish the source 
of the pus in every case of pyuria. This may at times be difficult, 
but the following data will be found of value in a differential diag- 
nosis : 

1. In disease affecting the renal parenchyma the amount of pus, 
as a rule, is small, except where a large abscess located in the kidney 
structure proper has burst into the pelvis of the kidney. 

In uncomplicated cases it is a comparatively easy matter to recog- 
nize the renal origin of the pus, as other constituents, such as renal 
epithelial cells, and tube casts, are usually present at the same time, 
and, as was noted in the case of renal epithelial cells, leukocytes 
are frequently found adhering to the tube casts, and at times appar- 
ently compose these entirely, when they are spoken of as pus casts. 
(See Casts.) In nephritis, according to Bizzozero, the number of 
pus corpuscles stands in a direct relation to the intensity and acute 
character of the morbid process, the greatest number being found in 
cases of acute nephritis, while in chronic forms their number is usually 
insignificant. Whenever in the course of a chronic nephritis large 
numbers of pus corpuscles appear, they may be regarded as indicating 
either an acute exacerbation of the disease or a complicating inflam- 
mation of some portion of the urinary tract. In such cases errors 
may be guarded against by observing the number and character of 
the epithelial cells present at the same time, when it will often be found 
that what at first sight appears as an acute exacerbation of a chronic 
process, judging from the number of pus corpuscles, is in reality a 
secondary pyelitis, ureteritis, or cystitis. 

In cases of simple renal hyperemia pus corpuscles never occur in 
notable numbers. 

2. In pyelitis the amount of pus may vary considerably, and at 
times even perfectly clear urine may be voided. This is prob- 
ably owing to the fact that the ureter of the affected side, if the dis- 
ease is unilateral, becomes obstructed temporarily, when suddenly 
large quantities may appear again. The diagnosis of pyelitis is 
often difficult, and should be based not only upon the condition of 
the urine, but upon the clinical symptoms as well. Very significant 
is the fact that the urine in pyelitis is usually acid. A careful exami- 
nation of the epithelial elements may also be of value, and should 
never be neglected. Bacteria in large numbers are generally present. 

In renal tuberculosis pus appears very early, but the amount may 
be extremely variable. Sometimes only a few leukocytes are seen, while 
at other times it may amount to one-fourth and even one-half of the 
urine by volume. As a rule the pyuria is constant, but cases are 
seen where for months and even years the urine may be almost 



566 THE URINE 

clear and the condition is much improved. It should be remem- 
bered, however, that the passage of apparently normal urine may 
merely indicate that the other ureter is blocked. 

When pyelitis is associated with nephritis it may at times be 
almost impossible to determine the origin of the pus; but if the rule 
set forth above is remembered, that in chronic nephritis the number 
of leukocytes is small, it is not likely that a pyelitis will be overlooked, 
particularly if the clinical symptoms are taken into consideration. 

Matters may become still more complicated when a cystitis is 
accompanied by a pyelitis or a pyelonephritis. Catheterization of 
the ureters should then be resorted to, and it is highly desirable that 
this most valuable method of diagnosis should become common prop- 
erty as soon as possible. Fischl regards the presence of cylindrical 
masses composed of pus corpuscles, formed in all probability in the 
papillary ducts, as highly characteristic of pyelitis. 

3. A pyuria referable to ureteritis can hardly be diagnosticated from 
the appearance of the urine, and in suspected cases catheterization of 
the ureters should be resorted to, which will probably throw light upon 
the question. 

4. In mild cases of cystitis pus may be altogether absent, while 
in the more severe forms its presene is constant. In cystitis the 
largest amounts, referable to disease of the urinary organs, are 
observed, and are exceeded only in those rare conditions in which 
a neighboring abscess has suddenly opened into the urinary passages. 

As the urine in cystitis is commonly alkaline, and always so in the 
more severe forms, the alkalinity being due to ammoniacal fermen- 
tation, it may happen, owing to the disintegrating action of the 
ammonium carbonate upon the pus corpuscles, that these may not 
be demonstrable with the microscope, and that a gelatinous mucoid 
sediment appears instead, which escapes from the vessel en masse 
when the urine is poured out. The chemical tests for pus, described 
above, must then be employed. 

5. In urethritis pus may be present in the urine in considerable 
amounts. The source of the pus is recognized by the fact that a 
drop may be manually expressed from the urethra, particularly in 
the morning upon awaking. Mucoid gonorrheal threads, — the 
" Tripperf aden" of the Germans, — which are largely composed of 
pus corpuscles, will almost always be detected in the urine in 
such cases. In order to distinguish between a simple urethritis 
and a urethritis complicated with cystitis, the urine should be ob- 
tained in two portions and allowed to settle. In simple urethritis 
affecting the anterior portion of the urethra the first specimen is 
cloudy, while the second one is clear. If the urethritis, however, 
has extended to the neck of the bladder, in the absence of cystitis, 
the first portion will, of course, be cloudy, while the second may 
present a variable appearance, being clear at times and cloudy at 



MICROSCOPIC EXAMINATION OF THE URINE 567 

others. This phenomenon is explained by the fact that a portion of 
the pus contained in the posterior portion of the urethra has found 
its way into the bladder. A cystitis may, however, be excluded by 
the acid reaction of the second specimen, and the fact that the latter 
is never so cloudy as the first. In cases of urethritis complicated 
with a purulent cystitis the second portion of the urine contains at 
least as much pus as the first, and usually more, owing to the fact 
that the pus (which is heavier than the urine) falls to the floor of the 
bladder, in which case also the last drop passed will often be found 
to be pure pus. The reaction of the urine, moreover, will then be 
generally alkaline. 

6. A sudden elimination of large quantities of pus with a urine 
which up to that time has presented a normal or nearly normal ap- 
pearance may almost always be referred to rupture of an abscess into 
the urinary passages. Exceptions to this rule have been noted in 
rare instances in which large amounts of pus suddenly appeared, the 
origin of which could not be demonstrated upon postmortem inves- 
tigation. Whether such a phenomenon, as v. Jaksch suggests, is 
dependent upon " unusual conditions favoring diapedesis" remains 
an open question. 

Enumeration of the Pus Corpuscles in the Urine. — In order to 
determine the relation existing between the degree of pyuria and 
albuminuria, as well as to watch the progress of an individual case, 
an enumeration of the number of pus corpuscles is at times neces- 
sary. To this end a specimen of the urine is thoroughly shaken 
and the number of corpuscles contained in one cubic millimeter 
ascertained with the aid of the hemocytometer (Simon's ruling). 
Dilution with a 3 per cent, solution of common salt is necessary if 
a preliminary examination has shown the presence of more than 
40,000 corpuscles per cb. mm. A dilution of five times is usually 
sufficient. 

Some of the results which have thus been obtained are extremely 
interesting. In cases of mild cystitis 5000 pus corpuscles are found 
on an average in the cubic millimeter; in cases of moderate severity 
from 10,000 to 20,000; while in severe cases 50,000 and even more 
may be seen. In one case of cystitis complicating carcinoma of the 
bladder Hottinger obtained 152,000 per cb. mm. In the presence of 
less than 50,000 a mere trace of albumin is found, and with 80,000 
to 100,000 only 1 pro mille is referable to this source. 1 

Red Blood Corpuscles, — The presence of red blood corpuscles in 
the urine, constituting the condition usually spoken of as hematuria, 
is observed only in pathological conditions, and is, in contradistinc- 
tion to hemoglobinuria (which see), a relatively common occurrence. 

1 R. Wunderlich, Ueber d. Werth d. Zahlung d. weissen Blutkorperchen im 
Harn, etc., Diss., Wiirzburg, 1885. 



568 THE URINE 

Urine containing blood corpuscles in notable numbers presents a 
color which may vary from a bright red to a dark brown verging 
upon black. Upon standing, a sediment of a corresponding color 
is obtained in which distinct coagula of variable size are at times 
seen. 

If the urine should contain only a small number of red corpuscles, 
however, no deviation from its normal appearance will be noted, and 
the diagnosis of hematuria can then only be made with the micro- 
scope, which should be employed in every case. The appearance of 
the red corpuscles varies greatly, being influenced especially by the 
length of time during which they have remained in the urine. In 
cases of hematuria of urethral or vesical origin it will be found that 
they have mostly retained their normal appearance fairly well, or 
have become crenated, when they may be recognized without diffi- 
culty. In cases, on the other hand, in which the corpuscles have 
remained in the urine for a longer time, as in hematuria of renal origin, 
the inexperienced will frequently be puzzled by the presence of bodies 
of the size of red corpuscles, or somewhat smaller, which are entirely 
devoid of coloring matter, and appear as faint, transparent rings, 
often presenting a double contour, and in which no nucleus can be 
discovered. These formations are red blood corpuscles from which 
the hemoglobin has been dissolved. They are spoken of as blood 
shadows. Chemical tests are rarely necessary, but may be employed 
if doubt should arise. 

Clinically it is, of course, all-important to determine the source of 
the blood. This may at times be accomplished without much diffi- 
culty by a urinary examination, but at other times it may almost be 
impossible, when the clinical symptoms and physical signs must be 
taken into consideration. 

1. Hematuria of urethral origin, due to urethritis, prostatitis, or 
traumatism incident to catheterization, for example, is a common event, 
and readily diagnosticated, as in such cases blood either escapes of 
itself from the urethra or it may be squeezed out manually. The 
last portion of the urine voided, moreover, will always be found free 
from blood, unless it is referable to disease of the neck of the bladder, 
when the blood appears only toward the end of micturition, or at 
least more markedly then than in the beginning. 

2. The diagnosis of vesical hematuria is not always easily made. 
It should be remembered, however, that the blood corpuscles here 
present a normal appearance, as has been mentioned, unless ammo- 
niacal decomposition is occurring in the bladder, in which case blood 
shadows are seen in large numbers. The blood, moreover, is less 
intimately mixed with the urine than in cases of renal hematuria, 
so that the corpuscles rapidly settle after the urine has been passed. 
Blood clots of an irregular form and considerable dimensions can 
only be of vesical origin. A careful examination for the presence 



MICROSCOPIC EXAMINATION OF THE URINE 569 

of any other morphological constituents whiclrmay be observed m 
urinary sediments, when considered in conjunction with the clinical 
symptoms, will usually lead to a correct diagnosis so far as the seat 
of the hemorrhage is concerned. Hematuria of vesical origin may 
be due to numerous causes, among which may be^mentioned hemor- 
rhagic cystitis, stone, tuberculous ulceration, malignant growths, papil- 
loma, traumatism, the presence of parasites, and, more rarely, rupture 
of varicose veins in the bladder. In determining the cause of the 
hemorrhage in a given case more reliance should be placed upon the 
clinical history and a direct examination of the bladder, than upon 
the urinary examination. 

3. In hematuria of ureteral origin characteristic blood coagula, 
corresponding in diameter and form to the ureters, are occasionally 
seen. Their presence, however, does not necessarily indicate that 
the blood has come from the ureters; more frequently the hem- 
orrhage will be found to be due to disease of the pelvis of the 
kidney. 

4. The diagnosis of hemorrhage into the pelvis of the kidney 
must be based upon the clinical symptoms taken in conjunction with 
the results of a urinary examination. In nephrolithiasis only a small 
number of red corpuscles is usually found, which is important from 
the standpoint of differential diagnosis. In renal tuberculosis hema- 
turia is one of the most important symptoms and not infrequently 
the first which attracts the attention of the patient. The amount 
is variable; sometimes the bleeding is microscopic, while in others 
almost pure blood is passed. It is usually intermittent, the periods 
of bleeding lasting from one hour to several weeks, the average being 
three days. Late in the disease it is usually less in amount, but apt 
to be almost continuous. As a rule the urine and blood are intimately 
mixed. Clotting, however, may occur in the bladder and the pelvis 
of the kidney. 

5. Hematuria of purely renal origin is of common occurrence, and 
may be due to numerous causes. In simple hyperemic conditions of 
the organs and in hemorrhagic nephritis the passage of smoky-looking 
urine containing blood corpuscles, usually in large numbers, is thus a 
fairly constant symptom. In chronic nephritis the number of the 
red corpuscles may be taken to indicate the intensity of the morbid 
process. Hematuria may also be due to renal abscess, renal tuber- 
culosis, malignant growths, stone, and, in rare instances, to aneurysm 
and embolism of the renal artery, thrombosis of the renal vein, papil- 
loma of the pelvis, etc. In the malignant forms of the acute infectious 
diseases, such as smallpox, yellow fever, malaria, etc., in scurvy, 
hemophilia, and purpura, in leukemia, filariasis, and distomiasis, 
renal hematuria is common. It is also observed in cases of poison- 
ing with turpentine, carbolic acid, cantharides, and has recently also 
been observed in several convalescents from typhoid fever while under 



570 THE URINE 

treatment with urotropin; the hematuria ceased with the discontinu- 
ance of the drug. 1 

6. Functional Hematuria. — An idiopathic form of hematuria has also 
been described, in which hemorrhage from the kidneys occurs with- 
out apparent cause. This is relatively common. Senator speaks of 
it as renal hemophilia. It has repeatedly led to errors in diagnosis 
and more particularly in connection with renal tuberculosis, as it 
also is usually unilateral. The amount of blood is very variable, 
sometimes only microscopic, at others excessive. I have seen three 
cases of this kind in which no lesion existed which could be made 
responsible for the hemorrhage. In all three the attacks of hematuria 
were associated with anachlorhydria, while normal values were found 
between the attacks. Two. of the patients were males, and undoubt- 
edly neurasthenics. The third was a hysterical, chlorotic female, in 
whom hematemesis, pulmonary hemorrhages, and melena were also 
at times observed. 

Hematuria of renal origin is usually recognized without much 
difficulty, as in such cases tube casts bearing red blood corpuscles, 
and at times apparently consisting of these altogether, as well as 
numbers of renal epithelial cells, will usually be found upon exami- 
nation. The blood, moreover, is intimately mixed with the urine, 
and the individual corpuscles have mostly lost their hemoglobin and 
appear as mere shadows. The clinical history should, of course, 
always be taken into consideration, especially in determining the pri- 
mary cause of the hemorrhage. 

Urine containing red blood corpuscles is always albuminous, so 
that it may sometimes be difficult to decide in a given case whether 
the albumin found is due solely to the presence of blood or whether 
the hematuria is complicated with an albuminuria per se. Fre- 
quently it is possible to arrive at some conclusion by comparing the 
amount of albumin with the number of the red corpuscles, the pres- 
ence of a large amount of the former in the presence of only a small 
number of the latter indicating that the albumin is not altogether 
due to the blood. At other times it is impossible to gain information 
in this manner, when the only expedient left is to determine the 
quantity of albumin and of iron separately, and to ascertain whether 
the amount of iron found is sufficient to combine with that of the 
albumin. As a rule, however, the presence of serum albumin, aside 
from that contained in the blood of the urine, may be inferred whenever 
tube casts are present, although the amount can only be estimated 
approximately in this manner. 

Tube Casts.— In various pathological conditions, and it is claimed 
even in health, curious formations are seen in the urine, which repre- 
sent molds of different portions- of the uriniferous tubules. To these 

1 Griffith, Milligan, and Forbes, Brit. Med. Jour., June 29, 1901. 



MICROSCOPIC EXAMINATION OF THE URINE 571 

the term tube casts or urinary cylinders has been applied. The term 
"tube casts/' however, is not altogether appropriate, as it is applicable 
to only one great division of such formations — i. e., to those con- 
sisting of a uniform, transparent, gelatinous matrix, to which other 
elements, such as epithelial cells, red blood corpuscles, leukocytes, 
and salts in a crystalline or amorphous form, may accidentally have 
become attached — the tube casts proper. 

From these the so-called "pseudocasts" must be differentiated, 
a pseudocast being characterized essentially by the absence of a uni- 
form matrix. Closely related apparently to the true casts are the 
so-called cylindroids — i. e., band-like formations which resemble the 
former in appearance, and like these may carry various morpho- 
logical elements. It is thus necessary to distinguish between true 
casts, pseudocasts, and cylindroids. Of these, the true casts are the 
most important. They may be divided into hyaline and waxy casts, 
the two forms being readily differentiated by the fact that the former 
readily dissolve in acetic acid, while the waxy casts are either not 
affected at all by this reagent, or, if so, at least not so rapidly. The 
latter, moreover, are more strongly refractive, to which property their 
waxy appearance is due; their color is slightly yellow or yellowish 
gray, while the hyaline casts are colorless and usually very pale and 
transparent. 1 

Mode of Examination. — Unless a urine can be examined within 
a few hours afer being voided, it is well to add a small amount of 
chloroform, so as to guard against bacterial decomposition. The use 
of conical glasses is unsatisfactory, and I find it more convenient 
to keep the urine in well-stoppered bottles. Preserved with chloro- 
form it will keep almost indefinitely. Where a centrifugal machine 
is available the specimen can, of course, be examined at once. As 
soon as a sufficient amount of sediment has been obtained, a few 
drops are spread on a slide and examined, uncovered, with a low 
power. It is essential, however, to make use of the flat mirror and 
to avoid a bright light. If this is borne in mind, no difficulty what- 
ever will be found in demonstrating even the most hyaline specimens, 
though they may be present in very small numbers. In many text- 
books on urinary analysis the writers speak of the difficulty attend- 
ing the search for hyaline casts, and the advice is frequently given 
to color the preparations with a drop of a dilute aqueous solution of 
iodopotassic iodide, or of some other staining reagent, such as gentian 
violet, picrocarmine, methylene blue, or osmic acid. This is unneces- 
sary if the directions just given are strictly followed. If a bright 
light is used, however, I am willing to admit that even the most 
experienced examiner may be unsuccessful in his search. 

1 Rovida, see J. Moleschott, Untersuchung. z. Naturlehre d. Menschen u. d. 
Thiere, 1867, vol. xi, I, p. 182. 



572 THE URINE 

For the preservation of mounted specimens the following method, 
devised by Kronig, may be employed, though I personally prefer to 
keep the urine itself and to mount a fresh specimen when necessary. 
A drop of the sediment, best obtained by centrifugation, is spread 
on a cover-glass and allowed to dry in the air. It is then placed 
in a 10 per cent, solution of formalin for ten minutes, rinsed in 
water, and stained for about ten minutes in a concentrated solution 
of Sudan III in 70 per cent, alcohol. The excess of stain is removed 
by immersion for one-half to one minute in 70 per cent, alcohol, 
when the specimen is counterstained with Ehrlich's hematoxylin, 
rinsed in water, and mounted in glycerin. Evaporation is guarded 
against by ringing the specimen with asphaltum. The tube casts 
are thus stained a more or less pronounced blue, the nuclei of the 
leukocytes dark blue, and any fatty granules or needles of fatty acids 
that may be present a bright red. 

I have obtained very satisfactory results by pouring a small amount 
of a 1 per cent, aqueous solution of eosin into one of the tubes of the 
urinary centrifuge, filling up with urine and then centrifugating. 
The supernatant fluid is poured off and the sediment mixed with 
Farrant's solution; the specimens are finally ringed with asphaltum 
and keep for a long time. The hyaline casts appear a delicate rose, 
while the fatty casts are a bright vermilion and the brown, granular 
casts a reddish brown. Adhering granules or cells are colored a 
bright red. 

Liebmann 1 recommends a mixture of 2 grams of methylene blue 
dissolved in 100 c.c. of a 10 per cent, solution of formalin. The 
urine is first centrifugated, the supernatant fluid is poured off, when 
a few drops of the reagent are poured on the sediment, and left a 
few minutes. The tube is filled with water, left for awhile for the 
salts to dissolve, then centrifugated again, when the formed elements 
are ready for microscopic examination. 

True Casts. Hyaline Casts (Plate XX). — Upon careful 
examination it will be seen that with rare exceptions the matrix of 
hyaline casts is not altogether homogeneous, as small granules may 
almost always be detected embedded in or adhering to the matrix. As 
these granules occur in greater or less numbers, hyaline casts are 
spoken of as being finely granular (Plate XX), coarsely granular, 
finely dotted, etc. Should true morphological elements be de- 
tected, the casts are termed blood casts, epithelial casts, or pus 
casts (Fig. 166). It would be better, however, to add the term 
hyaline in every instance, so as to distinguish them from pseudo- 
casts, which consist of these elements entirely, and lack a uniform 
matrix. It would thus be proper to speak of hyaline epithelial casts, 

1 Hospitalstidende (Copenhagen), July 30 to August 20, 1902. Abst. in Jour. 
Amer. Med. Assoc., September 20, 1902. 



PLATE XX. 





W 



% 



r/ 



m 



* j 




Casts. 

a, a, waxy casts; b, same, stained with eosin; c, c, c, hyaline casts; d, same, 
stained with eosin; e, e, e, brown granular casts; f, f, coarsely granular casts; 
g, epithelial cast; f, blood cast, stained with eosin. (Low-power picture, Leitz 3.) 



MICROSCOPIC EXAMINATION OF THE URINE 



573 



hyaline blood casts, etc., and to apply the collective term — compound 
hyaline casts — to these various subvarieties. 



Fig. 166. — Pus and epithelial casts. 



The nature of these various forms can probably always be made 
out without much difficulty, and even in those cases in which the 



M* 



m 






JP 










Fig. 167.— Pus cells from a urinary sediment. 



hyaline matrix is apparently concealed beneath cellular elements it 
will usually be possible, upon closer observation, to detect a fine 



574 THE URINE 

boundary line at some portion of the structure. Not infrequently 
also the end of the cast will be seen to be more or less distinctly 
hyaline. In others, again, a hyaline zone may be 'observed along 
the sides of a central organized thread, so to speak, this being fre- 
quently seen in specimens which are very broad and long. Should 
doubt arise, however, a drop of acetic acid is added to a drop of 
the sediment on the slide; the acid dissolves the hyaline matrix, 
the organized constituents are set free, and the differential diagnosis 
between a pseudocast and a compound hyaline cast is thus readily 
established. 

The length of hyaline casts varies greatly. It may scarcely exceed 
the breadth, on the one hand, while, on the other, although rarely, 
the casts may traverse the entire microscopic field. In breadth 
they vary between 0.01 and 0.05 mm. As a rule, the breadth of 
a cast is uniform throughout its entire length, but specimens are 
not infrequently observed in which one end tapers considerably and 
presents a spirally twisted appearance. This may be so marked 
that the entire cast appears transversely striated. It is generally 
supposed that this results from the adhesion of one end of the cast 
to the walls of a tubule, the lumen of which it does not fill, so that the 
free end becomes twisted in the downward course. A dichotomous 
branching of one end is also at times seen in very broad hyaline 
specimens. 

Fat globules are frequently found upon hyaline casts and are prob- 
ably derived from degenerated epithelial cells. When present in 
large numbers such casts are termed fatty casts. The globules are 
soluble in ether and are colored red by Sudan III. (See Tests for Fat.) 

Granules of melanin, indigo, and altered blood pigment may also 
at times be observed in casts. 

Regarding the mode of formation of the hyaline casts it is now 
thought that the matrix is essentially an inflammatory exudate, 
formed through the activity of the morbidly altered epithelial cells, 
and subsequently coagulated in the tubules. 

Brown Granular Casts. — These should not be confounded with the 
compound hyaline variety. They show no evidence of a hyaline 
matrix and on staining with eosin they are colored a deep brownish 
red. (Plate XX.) Unstained they appear brown. They are 
unquestionably composed of epithelial cells which have undergone 
degeneration, the residual material being then packed together in 
cast form. They are quite brittle and often not longer than they are 
broad. The true nature of. these small masses can be made out by 
staining with eosin, when it will be seen that they stain exactly like 
the larger pieces that have not yet broken down. 

The waxy casts may be divided into two groups — true waxy casts 
and amyloid casts; but as the latter are not necessarily indicative of 
the existence of amyloid degeneration of the kidneys, such a classi- 



MICROSCOPIC EXAMINATION OF THE URINE 575 

fication is of only theoretical interest. They are readily distinguished 
from the hyaline casts by the characteristics mentioned above — i. e., 
their higher degree of refraction, their yellow or yellowish-gray color, 
and the fact that they are either not attacked at all by acetic acid or 
only very gradually. Their appearance suggests a' much more solid 
object than the hyaline casts. As a rule, only small fragments are 
found, but in some instances very long casts are seen and occasionally 
I have found such long casts which were branching. Some of these 
casts at times present a peculiar knotty appearance. With eosin the 
waxy casts are colored a bright vermilion, while hyaline casts show 
only a pink color. Waxy casts may also contain cellular elements, 
crystals, and amorphous mineral matter; but, as a rule, such compound 
casts are not so commonly observed as are those of the hyaline variety. 

As has been stated, some waxy casts give the amyloid reaction — i. e., 
they assume a mahogany color when treated with a dilute solution of 
iodopotassic iodide, which changes to a dirty violet upon the addi- 
tion of dilute sulphuric acid. It should be remembered, however, 
that this reaction in casts does not necessarily indicate the existence 
of amyloid disease of the kidneys, as the reaction may be absent in 
this condition, and present where amyloid degeneration does not 
exist. This curious phenomenon is usually explained by assuming 
that such casts have remained in the uriniferous tubules for a long 
time, and have there undergone certain chemical changes analogous 
to the so-called " amyloid metamorphosis" of old precipitates of 
fibrin. Frerichs has pointed out that fibrin which has remained in 
the uriniferous tubules for a long time becomes denser and yellowish 
in appearance, which would explain the fact that these casts are only 
with difficulty attacked by acetic acid. 1 

The waxy casts, like the brown granular casts, are ultimately sup- 
posedly of epithelial origin. 

Before leaving this subject it should be stated that " cast-like" 
formations consisting entirely of amorphous urates are not infre- 
quently encountered in urines, and according to Leube 2 they may be 
obtained from any urine if it is concentrated in a vacuum at a tem- 
perature of 37° to 39° C. Students frequently regard such forma- 
tions as coarsely granular casts, an error which may be guarded 
against if the characteristics of hyaline casts set forth above are 
borne in mind. Such structures are not colored by eosin. 

Bacteria (in cases of infectious pyelonephritis), hematoidin, and 
granular detritus frequently occur grouped in a cast-like manner; 
their nature is readily ascertained, as in the case of the so-called 
urate casts just described. 3 

1 Rovida, loc. cit. Kobler, Wien. klin. Woch., 1890, vol. iii, pp. 531, 557, 
574 576 

2 Zeit. f. klin. Med., 1887, vol. xiii. 

3 Martini, Arch. f. klin. Chir., 1884, vol. xvi, p. 157. v. Jaksch, Deutsch. med. 
Woch., 1888, vol. xiii, Nos. 40 and 41. 



576 



THE URINE 



Pseudocasts, consisting of epithelial cells or blood corpuscles and 
fibrin, are not often found in urinary sediments. The epithelial 
pseudocasts are probably seen only in cases of desquamative nephritis, 
and, unlike true casts, are hollow, the epithelium of the uriniferous 
tubules being thrown off en masse. Blood casts consist of fibrin, 
within the meshes of which red corpuscles are found; these either 




<,,*•*> 



Fig. 3 68. — a and b, cylindroids 
from the urine in congested kidney. 
(v. Jaksch.) 



Fig. 169. — Mucous cylinders. 



present a normal appearance or occur as shadows, owing to the fact 
that their hemoglobin has been dissolved. They are seen whenever 
extensive hemorrhage has taken place in the renal parenchyma, and 
are more common than the epithelial pseudocasts. 

Cylindroids (Fig. 168) resemble hyaline tube casts somewhat in 
general appearance, but differ from them in being much larger and 



MICROSCOPIC EXAMINATION OF THE URINE 577 

band-like. Like true casts, they have a uniform breadth, and are 
often beset with crystals and cellular elements, such as leukocytes, 
red corpuscles, and epithelial cells. They are readily dissolved by 
acetic acid, thus differing from the mucous cylinders or pseudo- 
cylinders (Fig. 169) which may be observed in any* urine containing 
mucus; the latter probably never contain morphological or mineral 
constituents, and are never of uniform breadth throughout their 
ength. The cylindroids proper are undoubtedly of renal origin 
and closely related to true casts; formations are indeed not infre- 
quently seen in which a tube cast terminates in a cylindroid at one 
or both ends. 1 

Clinical Significance of Tube Casts. — Formerly the occurrence of 
tube casts in urine was held to indicate the existence of nephritis. 
This view has been abandoned, however, for the same reasons which 
led to the rejection of the idea that albuminuria invariably indicates 
Bright's disease (see above). 

The statement is frequently made in text-books that tube casts 
may occur in the urine of perfectly healthy individuals, following 
severe muscular exercise, cold baths, etc. — in short, stimuli which 
may cause the appearance of albumin in apparently normal individ- 
uals. It has been indicated elsewhere (see Functional Albuminuria), 
however, that such stimuli cannot be regarded as "physiological" 
in every instance, and the presence of tube casts in the urine similarly 
should be regarded as a pathological event. 2 This, however, does not 
invalidate the now generally recognized fact that a small number of 
hyaline and granular casts can be demonstrated in the centrifugated 
urine of many people who are to all intents and purposes in good 
health. 

It is not necessary in this connection to enumerate the various 
diseases in which cylindruria is observed, as they are the same as 
those which give rise to albuminuria; and just as a nephrangiogenic 
albuminuria is more frequently observed than a nephritidogenic albu- 
minuria, so also is the presence of tube casts in the urine more fre- 
quently due to circulatory disturbances than to nephritis. In every 
case in which tube casts occur in the urine it may be assumed that 
the accompanying albuminuria is, to a certain extent at least, of renal 
origin. 

Formerly it was thought possible to diagnosticate the character of the 
underlying renal disturbance from the type of casts found in the urine. 
This, however, is not the case. While generally speaking blood and 
epithelial cells are found in acute and granular casts in chronic pro- 
cesses, there are exceptions so numerous that it would not be safe to 

1 Bizzozero, loc. cit. Thomas, Arch. f. Heilk., 1870, vol. xi, p. 130. Pollak 
u. Torok, Arch. f. exper. Path. u. Pharmakol., 1888, vol. xxv, p. 87. 

2 Nothnagel, Deutsch. Arch. f. klin. Med., 1874, vol. xii, p. 326. Burkhart, 
Die Harncylinder, 1884. Fischel, Prag. Vierteljahrschr., 1878, vol. cxxxix, p. 27. 

37 



578 THE URINE 

follow such a rule. It is remarkable to see the large number and the 
many varieties of casts which may be found in the urine during the 
first twenty-four to forty-eight hours after anesthesia, and to observe 
how rapidly they may disappear, no evidence remaining whatsoever 
that the renal parenchyma has shortly before been seriously taxed. 

Cabot has recently pointed out the lack of correspondence between 
the clinical diagnosis of renal disease, as based upon urinary examina- 
tion and the pathological findings, and has given expression to what 
many clinicians have previously realized, viz., that neither the diag- 
nosis nephritis nor the type of the renal disturbance can usually be 
made with certainty in the laboratory. My own experience has led 
me to the conclusion that so far as cylindruria is concerned the con- 
tinued presence of hyaline and granular casts, especially of the dark- 
brown variety, is a symptom of greater gravity than the temporary 
occurrence of the other types. Hyaline casts per se are found under 
the most diverse conditions. Almost any renal disturbance, whether 
temporary or permanent, leads to their appearance. Their number 
is sometimes most remarkable, notwithstanding the fact that no per- 
manent renal damage has been done. Finely dotted and finely 
granular casts are generally present at the same time. 

As the granular cast is generally viewed as a hyaline cast which 
has been retained in the tubuies for a longer time, and, as a result, 
has undergone changes leading to its granular appearance, it might 
be inferred that in many temporary disturbances this type is riot 
found. In a general way this is true, but the occasional finding of 
granular casts only should not lead to the diagnosis of a chronic dis- 
turbance. They also can appear quite suddenly and disappear almost 
as rapidly. 

Epithelial casts and blood casts are met with in acute processes or 
in acute exacerbations of chronic processes. 

Waxy casts always indicate a chronic or, at least, a subacute pro- 
cess. The fatty casts described by Knoll and v. Jaksch "are most 
commonly associated with subacute or chronic inflammations of the 
kidney of protracted course, with a tendency to fatty degeneration 
of the renal tissue. Postmortem examination has shown that they 
form most frequently in cases of large white kidney. In some cases 
in which they were present, however, the organ was found to be more 
or less contracted; but when this was so, it was invariably far advanced 
in fatty degeneration/' (v. Jaksch.) 

It has been stated that from an examination of the renal epithelial 
cells it is often possible to determine whether an inflammatory pro- 
cess affecting the kidneys is at the same time complicated with de- 
generative changes. As a matter of fact, the cells found on the tube 
casts under such conditions no longer present a normal appearance, 
but are shrunken and atrophied, and in cases of fatty degeneration 
studded with fatty granules. 



MICROSCOPIC EXAMINATION OF THE URINE 



579 




/ 



The occurrence of pus casts presupposes the existence of suppura- 
tive inflammation in the kidneys, while the presence of only a small 
number of leukocytes on hyaline casts may be observed in the ordi- 
nary forms of nephritis, and particularly in the acute form. 

Cylindroids are present whenever hyaline casts are seen, and have 
essentially the same import. They are said to occur most frequently 
in the urine of children. 

So far as the constancy is concerned with which tube casts occur 
in the urine in nephritis, it is well known that in the chronic inter- 
stitial form of the disease, they, as well as albumin, are frequently 
absent for a long time, so that it may only be possible to make the 
diagnosis from the clinical history and the physical signs. It is a 
well-known fact, moreover, that pathological alterations of the kid- 
neys, particularly in men past middle 
age, are observed again and again i . .~ 
in the postmortem room, where a ; \ * 

previous examination of the urine 
showeed no evidence of the exist- 
ence of renal disease. In the acute 
and subacute forms of nephritis, as 
well as in the ordinary parenchy- 
matous form, tube casts are prob- 
ably always found, and it would 
further appear that acute circulatory 
disturbances affecting the renal par- 
enchyma quite constantly lead to 
both albuminuria and cylindruria. 

Within recent years attention has 
been repeatedly called to the occa- 
sional occurrence of cylindruria without albuminuria. Nothnagel 
first noticed this in a case of icterus. Liithje observed the same after 
administering salicylic acid and Stewart has drawn attention to its 
occurrence in the early stages of chronic nephritis. I have observed 
the same after the administration of ether. 

Spermatozoa. — Spermatozoa, for a description of which the reader 
is referred to the chapter on the Semen, are frequently observed in 
the urine of healthy adults, and are quite constantly met with in the 
first urine passed after coitus or nocturnal emissions, when their 
presence is, of course, of no significance (Fig. 170). 

In females semen may be found in the urine when the external 
genitals have been polluted during or after coitus, as well as in the 
exceptional cases in which connection has been effected by the urethra. 
From a medicolegal standpoint the discovery of spermatozoa in the 
urine of women may be of great importance, but otherwise it is with- 
out significance. 

In pathological conditions spermatozoa are not infrequently found 



©: 




Fig. 170.— Spermatic fluid, showing sper- 
matozoa, corpora amylacea, and lecithin 
corpuscles. 



580 THE URINE 

in the urine. In cases of obstinate constipation, owing to pressure 
of hard, scybalous masses upon the seminal vesicles, a partial evacu- 
ation of semen may occur. Horowitz has pointed out that a dis- 
charge of semen may be noted in cases of peri-urethral abscess with 
perforation into the ejaculatory ducts, giving rise to spermatocystitis , 
the condition being due to a tight stricture of the urethra with dila- 
tation beyond the constricted portion. I have observed a case of 
cystitis in which spermatozoa could almost always be detected in the 
urine. An operation revealed a tight stricture of the urethra and a 
sacculated bladder; the constant passage of semen was apparently 
owing to the irritating action of the ammoniacal urine. In the urine 
voided during and after epileptic and, more rarely, hystero-epileptic 
seizures spermatozoa may be found. Such an event is undoubtedly 
due to muscular spasm, and is identical in origin with the emission of 
semen observed so frequently in the death agony, and during strangu- 
lation. 

In certain spinal diseases semen may be found in the urine, and 
Fiirbringer relates a case in which, following fracture and dislocation 
of the vertebral column, with partial destruction of the middle dorsal 
cord, spermatorrhea associated with partial erection occurred thirty 
hours later, and continued until death, which took place after three 
days. 

More important is the loss of semen noted in cases of true sperma- 
torrhea due to venereal excesses or masturbation, when spermatozoa 
may be found almost constantly, and the diagnosis indeed will often 
be dependent upon such an observation. 

So far as the question of sterility in the male is concerned, reliance 
should not be placed upon an examination of the urine, but the semen 
should be obtained as soon as possible after ejaculation, and exam- 
ined as indicated elsewhere. 

Parasites. Vegetable Parasites. — It has been shown by numerous 
investigations that bacteria are always present both in the male and 
female urethra, and that they may at times gain entrance to the 
bladder. The weight of evidence, however, is in favor of the view 
that the urine intra vesicam is under normal conditions free from 
microorganisms, and that bacteria which may have found their 
way into the bladder are rapidly killed in healthy individuals. In 
every urine, on the other hand, that has been exposed to the air, 
bacteria are always present. Whenever, then, it is desired to deter- 
mine whether or not the urine of the bladder contains microorgan- 
isms, every precaution should be taken to guard against accidental 
contamination. To this end the following method should be em- 
ployed: If the patient is a male, he is instructed to hold his urine 
until a fairly large amount has accumulated. The glans is then 
thoroughly washed with soap and water, and further cleansed with 
cotton soaked in mercuric chloride solution (1 to 1000). The fossa 



MICROSCOPIC EXAMINATION OF THE URINE 581 

navicularis is also thorougly cleansed with the same solution. The 
urine is then voided under as great pressure as possible. The first 
portion (about 100 c.c.) is thrown away, and the second received in 
a sterilized vessel, when cultures should be made at once, agar or 
gelatin plates being inoculated with 1 or 2 c.c* of the urine. In 
the female the vulva is cleansed with soap and water, and the urethral 
aperture disinfected with bichloride solution. After then washing 
with sterilized water and drying with sterilized cotton the urine is 
evacuated through a sterilized metallic or glass catheter, and received 
in a sterilized vessel. Brown describes the method which is in use 
in Dr. Kelly's department at the Johns Hopkins Hospital as follows : 
The external urethral orifice being carefully cleansed with mercuric 
chloride solution, followed by sterile water, a sterilized glass catheter, 
whose external end is covered by a sterile rubber cuff, extending 
several centimeters beyond the end of the catheter, is introduced, 
the fingers of the operator being allowed to touch only the distal end 
of the rubber cuff. The urine is allowed to flow for a short time, 
when the rubber cuff is pulled off by traction on its distal end. A 
small amount of urine is then collected in a sterile test-tube, and 
the cotton plug immediately inserted. Brown states that an extended 
series of experiments with normal urines has shown that this method 
is absolutely reliable. 1 

Of the bacteria which may be found in every urine that has been 
exposed to the air, the Micrococcus urece is of special interest, as ammo- 
niacal fermentation is largely due to its presence. When fermenta- 
tion has commenced, it is readily recognized, occurring in almost 
pure culture upon the surface of the urine, mostly in the form of 
characteristic chains. The individual coccus is colorless and quite 
large, so that it may be mistaken by beginners for a blood shadow. 

It is a common error to infer from the occurrence of ammoniacal 
decomposition very soon after micturition that this process has already 
begun in the bladder. It should be remembered that urine may un- 
dergo fermentation, particularly in warm weather, shortly after having 
been voided, and especially if the vessel employed is not perfectly 
clean and the urine has been exposed to the air. The diagnosis of 
ammoniacal fermentation in the bladder should hence only be made 
when the presence of ammonia can be demonstrated in the urine 
immediately upon being voided. , ..p 

Under pathological conditions various pathogenic bacteria may be 
found in the urine. Their presence usually indicates the existence 
of definite changes in the renal parenchyma, although these changes 
are not necessarily of an inflammatory character. Pyogenic cocci 
are especially prone to settle in the kidneys, and there give rise to 
focal inflammations; but even in the absence of such lesions they are 

1 T. R. Brown, loc. cit. 



582 THE URINE 

frequently found in the urine. In all forms of infectious nephritis 
an abundant elimination of bacteria may generally be observed. 
Von Jaksch states that in erysipelas the bacteriuria and nephritis 
disappear, together with the cessation of the disease, and in various 
suppurative processes taking place in the body the specific bacteria 
disappear from the urine within twenty-four to forty-eight hours 
after evacuation of the pus. 

Most interesting observations on the occurrence of bacteria in the 
urine of nephritic patients have been reported by Engel: 31 cases 
were examined. In 16 the Staphylococcus albus and aureus were 
found, in 8 pyogenic streptococci, in 4 the tubercle bacillus, in 5 the 
Bacillus coli communis, and in 1 the typhoid bacillus, while negative 
results were obtained in only 2 instances. In the same series Engel 
also found a pyogenic coccus in 17 cases. This coccus was larger 
than the known forms; it could be stained according to Gram's 
method, and did not liquefy gelatin. Intravenous injections of large 
numbers of the organism caused nephritis in rabbits. 

In pneumonia and pneumococcus infections in general the corre- 
sponding diplococcus may be found, and in erysipelas and strepto- 
coccus infections streptococci. In scarlatina streptococci have been 
found in a large percentage of cases; the urine was then more often 
albuminous than non-albuminous. 

In cases of pyelitis the colon bacillus is very frequently met with. 
It is usually present in pure culture, but may be associated with other 
organisms, notably the Proteus vulgaris and staphylococci. These 
latter may, however, also be met with in pure culture. 

In renal tuberculosis the corresponding bacilli appear very early 
and are always present in the pus and debris. The search for 
them is usually very tedious, and small numbers only are found, but 
at times they are very numerous. To demonstrate their presence the 
urine is allowed to settle for twelve hours. Slides are prepared, which 
must be free from fat. To this end they are boiled for thirty minutes 
in a strong solution of caustic soda and then washed for an equal 
length of time in running water, after which they are wiped dry. 
Two drops of the sediment are placed on each one of six slides. They 
are placed on a frame some ten inches above a Bunsen burner, which 
is kept low, so as to ensure slow evaporation. When thoroughly dry 
they are fixed by passing through the flame of the burner and placed 
for five minutes in a 5 per cent, acid (HC1) alcohol to dissolve the 
urinary salts. After washing in water the specimens are then stained 
as usual. Using Gabbett's method they are stained for ten minutes 
with the carbol fuchsin solution and then decolorized with the acid 
methylene blue. If but little pus is present the urine may be cen- 
trifugalized. 

Using the above method Walker states that he could demonstrate 
tubercle bacilli in each case in which tuberculosis was afterward found. 



MICROSCOPIC EXAMINATION OF THE URINE 583 

In doubtful cases animal inoculation should be practised. The 
urine is received by the catheter into a sterile bottle, the first portion 
being allowed to escape. After twelve hours the supernatant fluid 
is poured off and the sediment drawn into a sterile hypodermic syringe. 
The material is injected into the subcutaneous tissue of the back of 
a guinea-pig. If tubercle bacilli are present tuberculosis should 
develop in from three to five weeks, but may occur even after two 
weeks. 

Intraperitoneal injections may also be practised, although one is 
more apt to lose the animals from incidental infections before tuber- 
culosis may become manifest. It is said that such secondary organ- 
isms may be eliminated by heating the material for ten minutes at 
60° C. The appearances seen at autopsy are very characteristic. 
The spleen, lymph glands, and liver show marked lesions. In cases 
where death occurs rapidly (in two weeks) miliary tubercles will be 
seen all over the liver and spleen, while the lymph glands are only 
moderately enlarged. In less active cases the lymphatic picture is 
most pronounced; axillary, cervical, and peritoneal glands are very 
much enlarged and the spleen may be transformed into one huge, 
caseating mass. 

On repeated occasions smegma bacilli have been mistaken for 
tubercle bacilli. They are quite common and especially met with 
in women; this, however, only in non-catheterized specimens. 
Greenbaum states that after thoroughly wiping the meatus and 
introducing a sterile catheter he never found them. 

In the male confusion with the smegma bacillus is less likely to 
occur, and if pains are taken to wash the glans and to irrigate the 
urethra, as advised by Young and Churchman, 1 it may be eliminated 
altogether as a disturbing factor. To this end the following technique 
is recommended: The foreskin, if present, is rolled back and held 
back by the patient. The glans is thoroughly scrubbed with soap and 
water. This must be done with great care, using very large amounts 
of water for the rinsing. An irrigator is filled with sterile water and 
the nozzle attached. This is made from a piece of small-caliber glass 
tubing with a circumference of a 15 F. sound and about seven and 
one-half inches long. The sharp edges of one end are rounded by 
fusing in the Bunsen flame. The other end is inserted into a piece of 
rubber tubing of the proper diameter to make a snug fit. The glass 
tube is pushed into the rubber tube about one inch, leaving about 
six and a half inches free. A rubber guard (conveniently made from 
one-half of a rubber ball) is fitted snugly over the rubber tubing near 
its end, about six and one-half inches from the fore end. The nozzle 
is connected with the tube of an ordinary irrigator, hung high enough 
to give a - good pressure, the patient being instructed to keep his 

1 Amer. Jour. Med. Sci., July, 1905, p. 52. 



584 THE URINE 

sphincter urethras closed during the procedure. The water is then 
allowed to flow, the glans and meatus well rinsed with it, and the nozzle 
gradually inserted and passed back to the triangular ligament (the 
tube is long enough to reach this), the stream flowing constantly during 
its insertion and withdrawal. A quart of irrigating fluid is used. 
(Young and Churchman.) 

Whether any reliable staining method exists whereby the smegma 
bacillus can be definitely distinguished from' the tubercle bacillus 
seems doubtful. Trudeau suggests staining in the usual way with 
carbol fuchsin, to decolorize with 25 per cent, nitric acid, then to 
wash and place the specimens for two minutes in 95 per cent, alcohol, 
and to counter-stain with blue. But he states that he does not find 
any method reliable in all cases and in doubtful cases advises inocula- 
tion of a guinea-pig. 

Of great interest is the frequent occurrence of the typhoid bacillus 
in the urine of typhoid-fever patients. Bouchard 1 in 1881 drew 
attention to the elimination of the bacillus through this channel, and 
stated that he was able to demonstrate its presence in 50 per cent, 
of his typhoid-fever cases. Other observers were less successful, but 
with improving technique and more general investigation a larger 
number of positive results is being obtained every year. 2 At the 
present time it may be said that the typhoid bacillus can be found in 
the urine of from 20 to 30 per cent, of all typhoid-fever patients. 
The organism usually appears in the second or third week of the 
disease, and may persist for months and even years. When present 
it usually occurs in pure culture, and often the bacilli are so numerous 
as to render cloudy a freshly voided specimen of urine. Symptoms 
of cystitis and marked renal involvement often occur, but in a consid- 
erable number of cases there are no indications of local disease. The 
elimination of the organism in the urine is of no prognostic significance, 
but is important from the standpoint of prophylaxis. Of special 
interest is the fact that the organism may at times be found in the 
urine, although the patient is not the subject of typhoid fever at the 
time. Brown 3 thus reports the case of a woman in whom a cystitis 
developed on the ninth day following an abdominal operation, and 
in whom it was thought that the typhoid bacillus was accidentally 
introduced by the catheter. The patient had had typhoid fever 
thirty-five years previously. Young 4 gives the history of a patient 
in whom cystitis developed during an attack of typhoid fever, owing 
to infection with the typhoid bacillus. The organism could still be 
demonstrated in the urine after several years. A double infection 

1 Rev. de med., 1881, p. 671. 

2 For an account of the literature, see T. R. Brown, "Cystitis due to the Typhoid 
Bacillus," etc., Med. Record, March 10, 1900. 

3 Loc. cit. 

4 " Chronic Cystitis due to the Bacillus Typhosus," Maryland Med. Jour., Nov., 
1901, p. 456. 



PLATE XXI. 













1 *,\ j " * 


/* 




i , 






ff^ 


<s?% -^ 


"*,- 






v.. 








1 r 



L SCHMIDT FEC. 



Urethral Discharge from a Case of Gonorrhea, showing Gonoeoeei Enclosed in 

Pus Corpuscles and. Lying Free in the Discharge- Stained with 

Methylene Blue. (Personal Observation.) 



MICROSCOPIC EXAMINATION OF THE URINE 585 

with the gonococcus subsequently occurred, and four months later 
typhoid bacilli and gonococci were both present in considerable 
numbers. Cystoscopic examination showed a chronic ulcerative 
cystitis. Two additional cases of chronic cystitis due to the typhoid 
bacillus are reported. 

The bacillus may be isolated and identified according to the usual 
methods. (See Blood and Feces.) 

In cases of paratyphoid fever the corresponding bacilli may be 
found in the urine. 

Gonococci may be found in urinary sediments enclosed in pus 
cells, and can be demonstrated by preparing smears and staining 
with a basic dye or with the eosinate of methylene-blue solution. 
In the so-called gonorrheal threads they can often be found years 
after the infection (Plate XXI). 

In cases of bubonic plague Kitasato's coccobacillus may be found 
in the urine. 

In cases of cystitis a great variety of microorganisms has been 
met with in the urine. Among the more important may be men- 
tioned the Staphylococcus aureus, albus, and citreus, streptococci, 
the Bacillus coli communis, the Bacillus pyocyaneus, the Bacillus 
typhosus, the Proteus vulgaris, the gonococcus, etc. In many cases 
of cystitis organisms are found, moreover, which are apparently non- 
pathogenic, and are capable of causing the formation of hydrogen sul- 
phide from certain sulphur bodies of the urine. (See Hydro thionuria. ) 

In conclusion, reference should be made to the occasional occur- 
rence of a form of bacteriuria which is not associated with any patho- 
logical process, and has hence been termed idiopathic bacteriuria. 
Of its causation and significance nothing is known, but it is pos- 
sible that in these cases a few bacteria enter the bladder either through 
the anterior rectal wall or are eliminated through the kidneys from 
the blood current. Finding a .suitable medium for their growth in 
the urine, they here multiply and may thus be constantly present. 
Of late, the Bacillus lactis aerogenes has been found in such a case. 
The diagnosis " idiopathic bacteriuria" should, of course, only be 
made if every possible source of contamination of the urine can be 
definitely excluded. 1 

Urines containing bacteria in large numbers are always cloudy, 
and usually present an acid reaction when voided unless cystitis 
exists at the same time. Attention is directed to their presence by 
the fact that such specimens cannot be cleared by simple filtration. 

Actinomyces kernels may be observed in the urine when the dis- 
ease in question has attacked the genito-urinary tract or when the 
organism has found its way into the urine from other organs. 

1 Roberts, "On Bacilluria," Trans. Internat. Med. Cong., London, 1881, vol. 
ii, p. 157. Schottelius u. Reinhold, Centralbl. f. klin. Med., 1886, vol. viii, p. 
635. Ross, Baumgarten's Jahresber., 1891, vol. vi, p. 360. 



586 THE URINE 

Yeast cells in large numbers are usually only seen in urines con- 
taining sugar. When a chemical examination has not been made 
their demonstration will be of importance, as suggesting the pos- 
sible existence of glucosuria. 

Molds are usually seen in old diabetic urines after alcoholic fer- 
mentation has taken place, but they may also occur, though far less 
frequently, upon the surface of putrid urines that have contained 
no sugar. 

The urinary sarcina which is at times met with is smaller than 
the sarcina of the gastric contents, but closely resembles it in appear- 
ance. It is of no clinical significance. 

Animal Parasites. — The organism which Hassal saw in a urine 
that had been "freely exposed to the air" and was alkaline, and 
which he termed Bodo urinarius, was in all probability an infusorial 
monad. Salisbury was the first to point out that the Trichomonas 
vaginalis of Donne may at times occur in the bladder, but he gave 
no detailed account of his cases. Kiinstler, Marchand, Miura, and 
Dock subsequently reported cases in which flagellate protozoa were 
found, and modern research leaves no doubt that the organisms 
described by these observers are identical with the trichomonas of 
Donne. In Miura's case the habitat of the parasite was the urethra, 
and an examination of the patient's wife revealed the presence of 
similar organisms in the vagina. Kiinstler's case was one of pyelitis 
following cystotomy. Marchand's patient had a fistula in the peri- 
neum following suppuration in the pelvis, of unknown origin; cystitis 
did not exist. Dock's case was associated with hematuria. 1 During 
the past few years I have seen the same organism in several cases, 
2 of which occurred in the practice of Dr. W. M. Lewis, of Balti- 
more. Most of them were women, and I have no doubt that the 
parasite found its way into the bladder from the vagina, where it 
could be demonstrated in 2 instances. Curiously enough a his- 
tory of hematuria was obtained from 4 patients. In 2 cases the 
urine contained blood at the time of the examination. In 1 case 
there was evidence of nephritis; cystitis did not exist. The number 
of the parasites was variable, and sometimes quite large. 

Balz observed innumerable amebas in the turbid urine of a girl the 
subject of phthisis, which he described as being of larger size than 
the Amoeba coli. Jiirgens found amebas in a patient suffering with 
a tumor of the bladder. Wijuhoff reports their presence in the urine 
in 4 cases. Posner cites 1 instance and Musgrave and Clegg another, 
the latter a case of hemorrhagic cystitis. 

In cases of bilharziasis the ova of the parasite (see Blood) are 
encountered in the urine together with blood. Sometimes the entire 
bulk of the urine is blood-tinged, but more often only the last few 

1 Amer. Jour. Med. Sci., January, 1896. 



MICROSCOPIC EXAMINATION OF THE URINE 587 

drops contain blood, and in these last drops the eggs of the parasite 
will also be found. In doubtful cases it is always best to examine 
this portion. The eggs are readily seen with a low power. (See 
Fig. 56.) 

Filaria embryos may be found in the urine in "cases of filarial 
chyluria. They should be looked for in the coagulum, a bit of 
which is teased out and pressed between two slides. 

Billings and Miller 1 have reported the possible occurrence of the 
Anguillula aceti in the urine, in cases in which the urine is collected 
in bottles that had contained old vinegar. The worm very closely 
resembles the Anguillula stercoralis. Stiles has made a similar 
observation. 

Echinococcus hooklets and fragments of cysts may also be found, 
and in rare instances ascarides find their way into the urinary pass- 
ages. Bothriocephalus linguloides (Leuckart) was found in the 
urine in a case occurring in Eastern Asia. Eustrongylus gigas is 
likewise found very rarely. Moscato records one case in which 
chyluria existed at the same time. In Clark's case, which was 
reported in the United States, the passage of the worm was accom- 
panied by hematuria. 

Tumor Particles. — Tumor particles are so rarely seen in the urine 
that a detailed account of their occurrence may be omitted, par- 
ticularly as it is seldom possible to base the diagnosis of tumor upon 
the presence of fragments in the urine, the clinical history and the 
physical signs being usually sufficient to reach a satisfactory diagnosis. 

Foreign Bodies. — Of foreign bodies which may be found in the 
urine may be mentioned particles of fat, fibers of silk, linen, and 
wool, etc.; in short, material the presence of which is owing to the 
use of unclean vessels for the reception of the urine. Fecal matter 
may be passed by the urethra; such an occurrence, of course, always 
indicates the existence of an abnormal communication between the 
bowel and the urinary passages. Hair derived from a dermoid cyst 
may similarly be found. In hysteria foreign bodies of almost any 
kind, such as hair, teeth, fish-bones, wood, etc., and even snakes and 
frogs, may be shown the physician as having been passed in the urine. 
I had occasion to examine "gravel" "passed" from time to time by 
a hysterical patient in large amounts, "every attack being accom- 
panied by agonizing pains shooting down into the lower abdomen;" 
the gravel upon examination proved to be mortar obtained from the 
cellar of the patient's house. 

1 Trans. Assoc. Amer. Phys., 1902, p. 161. 



CHAPTER VIII. 
TRANSUDATES AND EXUDATES. 

In health the so-called serous cavities of the body contain very 
little fluid, and quantities sufficient for analytical purposes can nor- 
mally only be obtained from the pericardial sac. In pathological 
conditions, on the other hand, large accumulations of fluid may be 
observed, not only in the serous cavities, but also in the areolar con- 
nective tissue, beneath the skin, and beneath the muscles. When 
due to circulatory disturbances, or a hydremic condition of the blood, 
such accumulations of fluid are spoken of as transudates, while the 
term exudates is applied to similar accumulations of inflammatory 
origin. 

Clinically, it is frequently difficult to distinguish between trans- 
udates and exudates, and large ovarian, pancreatic, and hydatid 
cysts, as well as cystic kidneys, may at times be mistaken for ascites. 
In such cases a careful chemical and microscopic examination of 
the fluid in question may be of value. Very frequently, moreover, 
it is possible only in this manner to determine the nature of the 
disease, and the free use of the trocar and, the aspirating needle in 
diagnosis cannot be too strongly advocated. 



TRANSUDATES, 

General Characteristics. — Transudates are usually serous in char- 
acter, when they present a light straw color; at times, however, owing 
to admixture of blood, they have a reddish tinge, and are then said 
to be sanguineous; in rare instances they are chylous. 

Specific Gravity. — The specific gravity varies somewhat according 
to the origin of the fluid, but is usually lower than that of serous 
exudates occurring in the same cavities— one of the most important 
points of difference between the two's kinds' of fluid. Thus, in acute 
pleurisy the specific gravity of the exudate is usually higher than 
1.020; and in chronic pleurisy, if an accumulation of pus exists at the 
same time, higher than 1.018, reaching even 1.030. In transudates 
into the pleural cavity, on the other hand, referable to circulatory 
disturbances, for example, as in cases of hepatic cirrhosis or cardiac 
insufficiency, the figures obtained are usually lower than 1.015. 
Transudates of peritoneal origin similarly present a specific gravity 



TRANSUDATES 5$9 

varying between 1.005 and 1.015, while that of exudates frequently 
reaches 1.030. 

As the chemical composition, in so far as the mineral constituents 
and extractives are concerned, is practically the same in both classes 
of fluid, the difference in the specific gravity appears to be essen- 
tially due to the amount of albumin present, viz., serum albumin and 
serum globulin. It may be demonstrated, as a matter of fact, that 
exudates contain far more albumin than transudates, the amount 
varying between 4 and 6 per cent, in the former, as compared with 
1 and 2.5 per cent, in the latter. The largest amounts of albumin 
in transudates are found in those of pleural origin, while in edema 
not more than 1 per cent, is usually present. 

Reuss suggests the following formula for the purpose of determining 
from the specific gravity the amount of albumin in transudates and 
exudates : 

E = f (5—1000) —2.8, 

in which E indicates the percentage amount of albumin and S the 
specific gravity taken by means of an accurate urinometer. 

Subsequent examinations have shown, however, that this formula 
is not applicable, since the amount of albumin is not strictly propor- 
tionate to the specific gravity. 

Since the use of Esbach's albuminimeter is totally insufficient for 
this purpose Strubell, Reiss, Strauss and Chajes, and Engel 1 sug- 
gest a refractometric examination, which depends essentially upon the 
amount of albumin present, but even with this method the results 
are not always satisfactory. Engel lauds it, however, nevertheless. 
An analysis of his data follows: 

Pleura. Abdomen. Pericardium. 

Nephritic transudates . . 1.3375 1.3374 1.3398 

1 . 04 per cent. . 98 per cent. 2 . 29 per cent. 

Cachectic transudates . . 1 . 3385 1 . 3382 1 . 3398 

1 . 59 per cent. 1 . 42 per cent. 2 . 29 per cent. 

Static transudates . . . 1 . 3392 1 . 3398 1 . 3405 

1 . 97 per cent. 2 . 29 per cent. 2 . 66 per cent. 
Pleuritic exudates . . . 1 . 3446 

4.89 percent. 

Peritoneal exudates 1 . 3445 

4 . 84 per cent. 

Pericardial exudates 1 . 3460 

5 . 64 per cent. 

The upper average figures indicate the refractometric coefficient, 
and the figures below the corresponding amount of albumin, as cal- 
culated from Reiss' tables. For a detailed description of the method 
the reader is referred to Reiss' paper. 2 

1 Strubell, Munch, med. Wochensch., 1902, p. 616. Reiss, Arch. f. exper. 
Pathol, and Pharmak, vol. li. Strauss and Chajes, ibid., vol. lii. Engel, Ber- 
lin, klin. Woch., 1905 ; p. 1364. 

2 Verhandl. d. 76 Versammlung deutscher Naturforscher u, Aerzte, Breslau, 
1904. 



59.0 TRANSUDATES AND EXUDATES 

The fact that transudates do not coagulate spontaneously in the 
absence of blood may further serve to distinguish them from exu- 
dates, in which a coagulum is frequently observed after standing 
for twenty-four hours. Not much reliance should be placed upon 
this point of difference, however, as exudates likewise do not always 
coagulate, and clotting of transudates in the presence of blood may 
take place within the body. 

Literature. — Reuss, Deutsch. Arch. f. klin. Med., vol. xxviii, p. 317. Rune- 
berg, ibid., 1884, vol. xxxiv, pp. 1 and 266; and Berlin, klin. Woch., 1897, No. 
33. Citron, ibid., 1897, p. 854; and Deutsch. Arch. f. klin. Med., vol. xlvi. 
Ranke, Mittheil. a. d. med. Klin. z. Wiirzburg, 1886, vol. ii, p. 189. 



CHEMISTRY OF TRANSUDATES. 

An idea of the chemical composition of the various forms of 
transudates may be formed from the following tables, taken from 
Hoppe-Seyler and Hammarsten, the figures corresponding to 1000 
parts by weight of fluid ; the specimens were taken from one individual : 

t,. -„ . Edema of 

Pleura. Peritoneum. t j ie f eet> 

Water 957.59 967.68 982.17 

Solids 42.41 32.32 17.83 

Albumin. . . . 27.82 16.11 3.64 
Ethereal extract 



Alcoholic extract 
Aqueous extract 
Inorganic salts 
Errors of analysis 



5.27 0.50 

) 3.71 

14.59 Q94 1.10 

luy4 9.00 

0.12 



Analysis of Hydrocele Fluid. 



Water 938.85 

Solids 61.15 

Fibrin (formed) . 59 

Globulins 13.52 

Serum albumin 35 . 94 

Ethereal extract 4 . 02 

Soluble salts 8.60 

Insoluble salts . 66 

Sodium chloride 6.19 

Sodium oxide 1 . 09 

Sugar and uric acid in small amounts are also, as a rule, found in 
transudates, and in one case of hepatic cirrhosis Moscatelli succeeded 
in demonstrating the presence of allantoin. v. Jaksch states that he 
has frequently been able to demonstrate the presence of urobilin in 
both transudates and serous exudates, even though red blood cor- 
puscles and blood-coloring matter in solution were absent. Stich 
also reports that in the ascitic fluid removed during life from a 
patient with hemorrhagic nephritis, urobilin was present. Peptone 
is never found; and Pajikull states that nucleo-albumin is not 
present in transudates of non-inflammatory origin. Hammarsten, 



EXUDATES 591 

together with Pajikull, could, however, demonstrate an albuminous 
substance in transudates which was regarded as a mucoid and which 
is present in exudates in small amounts only. It is rich in reducing 
substance and contains more nitrogen than the true mucins. 

Literature. — Moscatelli, Zeit. f. physiol. Chem., 1889, vol. xiii, p. 202. v. 
Jaksch, Zeit. f. Heilk., 1891, vol. xi, p. 440. Eichhorst, Zeit. f. klin. Med., 1881, 
vol. iii, p. 537. Stich, Munch, med. Woch., October 29, 1901. 



MICROSCOPIC EXAMINATION. 

Upon microscopic examination only a few isolated leukocytes 
and endothelial cells from the serous surfaces and undergoing fatty 
degeneration are usually seen. Mast-cells and eosinophilic leukocytes 
have been observed in the ascitic fluid in cases of myelogenous leu- 
kemia. Charcot-Leyden crystals were present at the same time. 
In cases in which the transudates have been confined for a long time 
plates of cholesterin are frequently found. They are especially 
abundant in hydrocele fluid. Amebas have been found by Miura 
in the ascitic fluid of a woman afflicted with an abdominal tumor; 
at the same time they were present in the stools. Leyden and 
Schaudin likewise met with ameboid bodies in the ascitic fluid 
obtained from two cases of abdominal tumor. The technique which 
should be employed in the microscopic examination of transudates 
is described below. 

EXUDATES. 

Exudates may be serous, serofibrinous, hemorrhagic, seropurulent, 
purulent, putrid, chylous, or chyloid. Of these, the seropurulent, 
purulent, and putrid types are manifestly of inflammatory origin, 
while in the case of the serous, serofibrinous, and hemorrhagic 
forms it may at times be difficult to determine whether the fluid 
represents a transudate or whether it is an exudate. A detailed 
chemical and microscopic examination may then be necessary. 

Serous exudates are clear, of a light straw color, and present a 
specific gravity which usually exceeds 1.018 (1.012 to 1.024). There 
is a large amount of fibrin and of albumin. If blood corpuscles are 
present in sufficient numbers to impart a distinct red color to the fluid, 
it is termed hemorrhagic; the color may then vary from a light 
pink to a dark red. On standing, even the purely serous exudates 
generally undergo a certain degree of coagulation, which becomes 
more marked in the presence of blood; exceptions, however, do 
occur. Most important is the microscopic examination of the 
exudates. Generally speaking, the same methods are here employed 
as in the case of the blood, but the interpretation of the findings is 



592 TRANSUDATES AND EXUDATES 

not always easy. This is largely owing to the fact that the leuko- 
cytes often show evidence of degeneration, and that the fluid may 
contain endothelial cells in addition to the morphological elements 
of the blood, which further increases the difficulties attending a 
proper classification. (See Pus.) The principal point at issue in the 
study of the cellular elements of exudates is the question as to the 
predominance of either lymphocytes or the polynuclear elements of 
the blood. Widal and his collaborators, more especially, have pointed 
out that whereas in exudates of non-tuberculous, acute inflammatory 
origin the polynuclear neutrophilic leukocytes predominate, the lym- 
phocytes prevail in the chronic tuberculous forms. His observations 
have, on the whole, been confirmed by numerous investigators, and 
the importance of cytodiagnosis in pleuritic effusions more especially 
is now well established. From the available data we may formulate 
the following conclusions: In the very earliest stages of tuberculosis 
involving the serous membranes there is found a variable number of 
neutrophilic leukocytes in addition to lymphocytes and endothelial 
cells. Very soon, however, they diminish and in the later stages the 
lymphocyte is by far the predominating cell, while the neutrophilic 
elements are present only in very small numbers. Generally speaking 
the percentage of lymphocytes in tuberculous pleurisies ranges from 
50 to 98, increasing as the disease continues. 

In pleuritic effusions due to the pneumococcus and to streptococci 
during the serous stages, the neutrophilic leukocytes far outnumber 
the lymphocytes. (Average in postpneumonic cases 71.7: variations 
from 58 to 92.5 per cent.) 1 In the pneumococcus cases, moreover, 
it is common to meet with large numbers of endothelial cells, some- 
times containing polynuclear leukocytes and red cells in their interior. 

In cases of traumatic and aseptic pleurisy, in association with 
diseases of the heart and kidneys, large endothelial cells are seen which 
often present most grotesque appearances, occurring either singly or 
in groups of two, three, four or more;'while the occurrence of large 
numbers of such cells has been regarded as characteristic of transu- 
dates, Carter has shown that in these cases also there may be a lym- 
phocytosis of from 86 to 100 per cent. ; so that confusion may arise in 
differentiating these cases from tuberculous pleurisy. The low spe- 
cific gravity — average about 1.008 — and the small amount of fibrin 
and albumin in the transudates will, however, aid in arriving at a 
conclusion. 

French writers also describe a pleural eosinophilia in which large 
numbers of eosinophilic cells — 6 to 54 per cent. — are found in the 
effusion, while in the circulating blood their number is not increased. 
Ravaut reports 4 cases of this kind. In 1 the effusion occurred 
secondarily in the course of syphilis; in the second in a case of typhoid 

1 H. S. Carter, Med. News, October 1, 1904 



EXUDATES 593 

fever; the third was a case of phthisis, while in the fourth no diagnosis 
was made. I have recently seen a case of this kind (probably tuber- 
culous) with 10 per cent, of eosinophiles, 4 per cent, neutrophils, 83 
per cent, of small mononuclears, and 3.4 per cent, .of large mononu- 
clears in the exudate, and 3.5 per cent, of eosinophiles, 42 per cent, of 
neutrophiles, 36 per cent, of small mononuclears, and 18 per cent, of 
large mononuclears in the blood. 

Carter 1 reports 2 cases of pleural effusion, referable to pistol-shot 
wounds of the chest walls, in which the eosinophiles numbered 70.2 
and 87.8 per cent., respectively. 

Mast-cells are rarely seen in pleuritic effusions, and it has been 
observed that their granules are then quite readily soluble in water, 
so that they cannot be demonstrated with aqueous solutions of the 
usual dyes. Wolff notes a case in which the mast-cells constituted 
about 10 per cent, of the total number of leukocytes. 

Whether or not the conclusions which have been reached regard- 
ing the meaning of the prevalence of certain cell forms in pleural 
effusions can be directly applied in the case of ascitic fluid remains 
to be seen. From the available data it appears that the indications are 
not as direct. But generally speaking endothelial plaques control the 
picture in ascites of mechanical origin, while lymphocytes predomi- 
nate in tuberculous peritonitis and in peritoneal carcinoma. The 
occurrence of large vacuolated cells is suggestive of a cyst accompanied 
by ascites (ovarian cyst). The same is true of the cytological study of 
joint effusions. Widal reports that in 3 cases of acute rheumatism 
he found polynuclear leukocytes in the serous exudate, while they 
were absent in traumatic cases of arthritis. As the result of an 
examination of 30 hydroceles Marchetti 2 concludes that lymphocyte 
and epithelial cells predominate without exception. 

Of the cytological findings in the cerebrospinal fluid a detailed 
account will be given later. 

Generally speaking the cytological factor does not seem to depend 
so much upon the anatomical localization of the morbid process as 
upon its duration and the character of the pathogenic agent. An 
acute process (pneumococci, streptococci) call forth a lymphocytosis 
of brief duration, which is followed sooner or later by a granulocytosis, 
while a less intense stimulus, and one acting more slowly (tubercle 
bacillus) leads to a persistent lymphocytosis. The possibility that a 
stimulus of the latter order may act with undue virulence and 
intensity, and that one of the first type may be exceptionally mild 
and delay the occurrence of granulocytosis, should, however, be 
borne in mind. 

Very important also is the study of the cellular elements which 

1 Med. News, October 1, 1934. 

2 Gaz. d. Ospedal. e. d. clin., 1904, No. 94. 

38 



594 TRANSUDATES AND EXUDATES 

are found in serous exudates in cases of malignant disease of the 
serous membranes. Difficulty may here be encountered in the in- 
terpretation of the cellular findings, for on the one hand it is often 
difficult to distinguish the endothelial cells from leukocytes, as they 
take on phagocytic activity and often present the most bizarre 
forms. The nucleus, which is normally centrally located, takes up 
an excentric position, and enclosed within the cell we may find leu- 
kocytes and red cells. On the other hand, it is impossible by simple 
inspection to distinguish normal endothelial cells from cancer cells. 
In cases of doubt it is well to ascertain whether the epithelial ele- 
ments give the glycogen reaction and to hunt for the presence of 
mitosis. Qunicke has pointed out that normal endothelial cells 
do not contain glycogen, and that a marked iodine reaction is very 
suggestive of carcinoma. Wolff, however, suggests that this test 
is probably not specific, and cites two instances in which he obtained 
a positive glycogen reaction, although a tumor did not exist. More 
important probably is the presence of mitoses. In non-malignant 
exudates epithelial cells never present evidence of mitosis, while in 
cases of tumor they may be found. Rieder regards their occurrence 
as pathognomonic of malignant disease. Commonly the mitosis is 
atypical; the division of the nucleus is not followed by a division of 
the cell; the chromosomes are short and show no polar or equatorial 
arrangement. 

In cases of neoplasm Quincke has also drawn attention to the 
occurrence of large numbers of fat droplets in the fluid, which may 
attain a diameter of from 40 to 50 fi. At times, however, the fat 
droplets are so small and so numerous as to give a chylous appear- 
ance to the exudate. At other times a similar appearance is due to 
the presence of minute albuminous granules, which may be distin- 
guished from fat by their insolubility in ether and the fact that they 
are not stained with the common fat dyes, such as Sudan, scarlet-R, 
and alkanin. The occurrence of numerous fatty acid crystals, 
arranged in groups, should also excite suspicion of a neoplasm. 

Should bits of tissue be obtained, a positive diagnosis of malig- 
nant disease may, of course, be made by the usual methods. Such 
particles should be placed at once in absolute alcohol or formalin. 

Crystalline elements are not usually seen in serous or hemorrhagic 
exudates; at times we meet with platelets of cholesterin. 

Technique. — In every case the fluid should be examined as soon 
after puncture as possible; if this cannot be done at once, coagula- 
tion may be prevented by the addition of sodium citrate. The 
material is then placed in the ice-box until a sediment has collected 
or this may be obtained at once by centrifugation, new portions of 
fluid being repeatedly used and the sediments combined. Cover- 
glass preparations may then be conveniently made, or smears on 
slides exactly as in the case of blood, care being taken to do as little 



BACTERIOLOGICAL EXAMINATION OF EXUDATES 595 

injury to the cellular elements as possible. The smears should be 
very thin, so that the specimens will dry rapidly and but little 
chance is given for the cells to contract beyond their usual size. 
Subsequent treatment will depend upon the special points which are 
to be elicited. Unfortunately the leukocytes are often much changed, 
so that their classification may be attended by considerable diffi- 
culties. The polynuclear elements may appear mononuclear and 
not infrequently the neutrophilic granules can no longer be demon- 
strated. (See Pus.) For this reason the triacid stain is not to be 
recommended for routine work; the eosinate is much better and 
will furnish as satisfactory results as can be obtained with a panoptic 
dye. Successive staining with eosin and methylene blue sometimes 
gives better results than a polychrome dye. Care should be had not 
to diagnosticate eosin ophilia from the fact that cell granules are 
stained red, as the neutrophilic granules of degenerating cells are com- 
monly amphophilic, viz., they stain both with acid and neutral dyes; 
account must be taken of the size of the granules and the general 
structure of the cell. To differentiate pseudolymphocytes from true 
lymphocytes, Pappenheim's methyl-green pyronin may be employed, 
though it is not absolutely specific; still it will be found that even 
though the protoplasm of other cellular elements may take the red 
color of the pyronin, the intensity is distinctly less than in the case 
of the lymphocytes proper. 

Pappenheim's Method. 1 — The stain is composed of a concentrated 
aqueous solution of methyl green to which pyronin is added until the 
solution just begins to turn blue viz., about 1 part of pyronin for 
3 to 4 parts of methyl green. Stained in this manner the basophilic 
protoplasm of the lymphocytes is colored a fine dark carmine red, while 
the protoplasm of all other cells is stained a more or less pale brownish 
or reddish yellow, or remains colorless. Pappenheim regards this 
stain as essentially specific for the lymphocytes, but admits that it 
also stains in a similar manner the young erythroblasts that are poor 
in hemoglobin. The difference can be recognized from the character 
of the nuclei and the fact that the margin of the lymphocytes very 
commonly appears shaggy, while that of the erythroblasts is smooth 
and homogeneous. 

To study mitosis, hematoxylin and eosin may be employed, or 
the Romanowsky method in one of its various modifications. 

The glycogen reaction is demonstrated as in the case of the blood 

BACTERIOLOGICAL EXAMINATION OF EXUDATES. 

In a measure the bacteriological examination of exudates has been 
supplanted by the cytological study, as outlined above; especially as 

1 Virchow's Archiv, 1899, vol. clvii. 



596 TRANSUDATES AND EXUDATES 

the bacteriological examination has been notoriously unsatisfactory 
in the most important group of effusions, viz., in those of tuberculous 
origin. It is now known that all exudates gradually become free 
from bacteria, even though at first they may have been caused by 
bacterial activity. As a result it is no longer justifiable to conclude 
that a process is tuberculous because bacteriological examination of 
the exudate has given no positive result. If it is desired to cultivate 
organisms that may be present, it is well to make a bouillon culture 
in every case so as to eliminate the bactericidal properties of the 
exudate as much as possible. In any event it is well to centrifugate 
the fluid in a sterile tube and to use the sediment for inoculations. 
The organisms which are most likely to be encountered are the pneu- 
mococcus, the various staphylococci, streptococci, and more rarely 
the colon bacillus and the typhoid bacillus. 

Inoscopy. 1 — Jousset recommends the following procedure for the 
purpose of demonstrating tubercle bacilli in exudates: The fluid is 
allowed to clot spontaneously or by adding a little horse serum. 
The clot, which is supposed to contain most of the organisms, is pressed 
out, torn into fragments, and placed in about 10 c.c. of a digestive 
mixture of the following composition: pepsin, 1 to 2 grams; glycerin, 
10 c.c; 40 per cent, solution of hydrochloric acid, 15 c.c; sodium 
fluoride, 3 grams ; water, 1000 c.c The material is left in the incubator 
for three to four hours, then centrifugalized and smears prepared from 
the sediment and stained as usual. Jousset claims to have obtained 
very good results in this manner, while others are less enthusiastic 

More recently Zebrowski 2 has suggested the following method as 
more likely to lead to satisfactory results: Coagulation of the fluid 
is prevented by the addition of an equal volume of a 0.5 per cent, 
solution of sodium fluoride. The mixture is set aside in a cool place 
until the following day, when it is thoroughly centrifugated and 
smears made from the sediment and stained as usual. 

With this method Zebrowski claims to have found tubercle bacilli 
in 83 per cent, of secondary and 55 per cent of primary pleurisies. 

More satisfactory than either method possibly is the animal 
experiment, to which end a large quantity of the fluid is centrifugalized 
and the sediment injected into the peritoneal cavity of a guinea-pig, 
as in the case of the urine (which see). 

Literature. — Widal and Ravaut, "Cytodiagnostique des epanchements sero- 
fibrineux de la plevre," Trans. XIII Internat. Med. Cong. Paris, 1900. Barjou 
and Cade, "Etudes cytol.," etc., Arch. gen. d. med., August, 1902. Gulland, 
"Cytodiagnosis," etc., Scott. Med. and Surg. Jour., June, 1902, p. 490. A. 
Wolff, "Transudates and Exudates," Zeit. f. klin. Med., 1902, vol. xxii, Heft 5 
u. 6. Quincke, Deutsch. Arch. f. klin. Med., 1882, vol. xxx, pp. 369 and 580. 
Rieder, ibid., 1895, vol. liv, p. 544. 

1 La semaine m6d., 1903, No. 3. 

2 Deutsch. med. Woch., September 7, 1905. 



CHEMISTB Y OF EX VBA TES 597 

CHEMISTRY OF EXUDATES. 

According to Moritz, an albumin is found in exudates that can 
be precipitated with acetic acid and which is absent in transudates. 
He regards this as serum globulin which has undergone a change as 
a result of the inflammatory process. According to Matsumoto, on 
the other hand, the substance in question represents a mixture of 
fibrinoglobulin, euglobulin, and a small amount of pseudoglobulin ; 
in the filtrate, however, there is also some fibrinoglobulin (fibrinogen) 
and euglobulin. He suggests that this last circumstance is probably 
referable to the small amount of salt in exudates and that in the first 
instance the pseudoglobulin is probably carried down mechanically. 

More recently Umber has studied the body in question and arrived 
at the conclusion that it belongs to the mucins. To its presence 
the mucinous character of such fluids is due. It is precipitated by 
the addition of acetic acid and is insoluble in an excess of the reagent 
unless the acid is present in great concentration. The body has 
markedly acid properties and is not coagulated by heat. It differs 
from the known mucins in the presence of a very small amount of 
reducing substance, which can only be demonstrated by special 
methods. It contains about 14 per cent, of nitrogen and no phos- 
phorus. In neutral and feebly acid solution the substance does not 
coagulate (thus differing from the globulins). The same body appa- 
rently was found by Salkowski in an exudate into the hip-joint. Umber 
calls this substance serosamucin. Its amount is less than 0.5 per cent. 
According to Umber and Stahelin the serosamucin is essentially 
found in exudates referable to inflammatory processes or associated 
with new growths. In transudates, as Runeberg already pointed 
out, only a very slight turbidity results upon the addition of acetic 
acid, and not in all cases, moreover; so that a well-marked reaction, 
viz., a marked precipitation upon the addition of acetic acid to the 
point of a distinctly acid reaction, may be regarded as a valuable 
sign in the diagnosis between transudates and exudates. I append 
some of the results obtained by Umber: 

Ascites. 

No. of cases. Serosamucin. 

Hepatic cirrhosis 6 

Hepatic cirrhosis with chronic nephritis and phthisis 1 

Nephritis 1 

Mitral disease 3 

Pleural Exudates. 

Degeneratio cordis and nephritis 2 

Myocarditis 1 

Hepatic cirrhosis 1 

Lymphosarcoma (pleura intact postmortem) 1 

Carcinoma mammse with pleural metastases 1 + 

Tuberculosis of pleura 1 + 

Pleuritis exsudativa acuta 1 + 

Pleuritis and pericarditis 1 + 



598 TRANSUDATES AND EXUDATES 

For the isolation of serosamucin see Umber's paper (see Literature 
below.) 

Of the common albumins we meet with traces of fibrinogen and 
with fairly large amounts of globulin and serum albumin. Their 
percentage may at times not appear so very large, but considering 
the large amount of fluid and the rapidity with which it may accumu- 
late it is clear that the loss of nitrogen to the body in this form may be 
very considerable. Umber showed that in one of his cases 5000 grams 
of albumin representing about 15,000 grams of muscle tissue were 
lost within a year. 

In addition to the serosamucin and the common albumins men- 
tioned, some exudates may possibly also contain small amounts of a 
a nucleo-albumin, as is suggested by the findings of Pajikull. Should 
ovarian cysts have ruptured into the peritoneal cavity, we may fur- 
ther find both pseudomucin and paramucin (which see). 

Of interest further is the fact that Umber succeeded in demonstrat- 
ing the existence of autolytic processes in exudates. He found both 
albumoses and mono-amino acids, viz., leucin and tyrosin. 

Coriat has reported a case of polyneuritic delirium, in which pleurisy 
with effusion developed. In the effusion he could demonstrate a 
peculiar albuminous substance, which he regards as identical with 
Bence Jones' albumin; in the urine this substance could not be 
found. 

Literature. — Pajikull (Swedish ref. by Hammarsten : Jahresber. f . Thierchem., 
1893). Moritz, Munch, med. Woch., 1902, No. 42. Matsumoto, Deutsch. Arch., 
1902, vol. lxxv, p. 409. Stahelin, Miinch. med. Woch., 1902, No. 34. F. Umber, 
Zeitsch. f. klin. Med., 1903, vol. xlviii, p. 364. Coriat, "The Occurrence of the 
Bence Jones Albumin in a Pleuritic Effusion," Amer. Jour. Med. Sci., 1903, vol. 
cxxvi, p. 631. 

Pus. 

General Characteristics of Pus. — If pus, which usually pre- 
sents a color varying from yellowish gray to greenish yellow, is 
allowed to stand for a time, a liquid gradually appears at the top, 
and increases in amount until it is finally possible to distinguish 
two distinct layers, the one above — the pus serum; the other at the 
bottom — the pus corpuscles. Upon the number of the latter the 
consistence as well as the specific gravity of the pus is dependent. 
This may vary between 1.020 and 1.040, with an average of 1.031 
to 1.033. Fresh pus has always an alkaline reaction, which may 
become neutral or slightly acid upon standing, owing to the develop- 
ment of free fatty acids, glycerin-phosphoric acid, and lactic acid. 
The color of pus serum may be a light straw, a greenish or a brownish 
yellow. 

Chemistry of Pus. — The chemical composition of pus serum 
and pus corpuscles may be seen from- the following tables : 



CHEMISTRY OF EXUDATES 599 

Analysis of Pus Serum. 

I. II. 

Water 913.70 905.65 

Solids 86.30 94.35 

Albumins 63.23 77.21 

Lecithin : . . . . f.50 0.56 

Fat 0.26 0.29 

Cholesterin 0.53 0.87 

Alcoholic extract 1.52 0.73 

Aqueous extract 1 1 . 53 6 . 92 

Inorganic salts 7 . 73 7 . 77 

Analysis of Pus Corpuscles. 

I. II. 

Nuclein 342.37) 

Insoluble matter 205 . 66 \ 673 . 69 

Albumins .137.62] 

Lecithin) i iq qq (75.64 

Fat j i4d -^ 1 75.00 

Cholesterin 74.00 72.83 

Cerebrin 51.99) 1n9 fi4 

Extractives 44.33) w*.** 

Albumoses are usually present, and are derived from the pus cor- 
puscles. Leucin and tyrosin are likewise frequently met with in the 
pus of old abscesses; and fatty acids, urea, sugar, glycogen, biliary 
pigments and acids (in catarrhal jaundice), acetone, uric acid, xanthin 
bases, cholesterin, etc., have occasionally been observed. 1 

Microscopic Examination of Pus. Leukocytes. — If a drop of pus 
is examined with the microscope, it will be seen to contain innumer- 
able leukocytes, many of which in perfectly fresh pus exhibit ameboid 
movements. The cells in question are usually almost altogether of 
the neutrophilic variety, and it may be questioned whether the lym- 
phocytes ever occur in true pus. Even in cases of lymphatic leukemia 
the predominating cell in abscesses is the polynuclear leukocyte or 
its degeneration forms. Mononuclear elements with basophilic pro- 
toplasm, however, are also met with, notably in the more chronic cases, 
but it is likely that they are derived from the connective-tissue cells 
and are not of hematogenic origin. Eosinophiles are only seen in pus 
under certain definite conditions, as in gonorrhea (see below), and 
mast-cells also are quite uncommon. 

In pus that is not perfectly fresh it is usually not possible to dem- 
onstrate the presence of neutrophilic granules. In such cells, more- 
over, we commonly meet with fragmentation of the nucleus, asso- 
ciated with marked pyknosis. This was first noted by Ehrlich in 
a case of hemorrhagic smallpox and in various exudates, and has 
subsequently been described by Michaelis and Wolff. The degenera- 
tion may proceed to fragmentation of the entire cell with the conse- 
quent formation of mononuclear neutrophilic forms (Ehrlich's pseudo- 

1 M. Pickardt, "Z. Kenntniss d. Chemie path. Ergiisse," Berlin, klin. Woch., 
1897, p. 844, 



600 TRANSUDATES AND EXUDATES 

lymphocytes). On the other hand, a type of degeneration is seen 
in which the nucleus does not become pyknotic, but swells to a large 
size and stains rather faintly with basic dyes. In such cells the proto- 
plasm appears as a narrow rim and the impression is gained as though 
the cell were in reality a leukocyte; if at the same time the granules 
have been lost, the differentiation may indeed be impossible, unless 
transition forms exist between the normal polynuclear neutrophile 
and the type in question. 1 

Owing to resorption cf water from accumulations of pus of long 
standing, such material finally assumes a caseous aspect, and the 
leukocytes will be seen to have greatly diminished in size, and to 
have assumed an angular, shrunken appearance; it is then hardly 
possible to demonstrate the presence of a nucleus, even after the 
addition of acetic acid. 

It is noteworthy that in cases of hepatic abscess referable to 
Amoeba coli it is seldom possible to demonstrate any normal leuko- 
cytes, and it will be seen that under such conditions the pus consists 
almost altogether of granular and fatty detritus, while in liver 
abscesses due to other causes the leukocytes usually present a fairly 
normal appearance. 

Mast-cells are only exceptionally seen in pus. 

Giant Corpuscles. — So-called giant pus corpuscles, measuring at 
times from 30 fJ. to 40 fJ. in diameter, have been observed in abscesses 
of the gum, hypopyon, and in the contents of suppurating ovarian 
cysts, but they do not appear to have any special significance. Upon 
careful examination these bodies will be seen to contain one oval 
nucleus, usually located eccentrically within the cell, and from one to 
thirty or even forty pus corpuscles. 2 

Detritus. — Fatty and albuminous detritus in variable amount may 
be observed in every specimen of pus, and increases with the length 
of time that it has been confined within the body. The same holds 
good for the presence of free nuclei, which were formerly regarded 
as young pus corpuscles, but which have now been definitely recog- 
nized as originating during the disintegration of the corpuscles. 

Red Corpuscles. — Red blood corpuscles in variable numbers are 
usually seen in every specimen, their appearance depending upon the 
length of time they have been confined. Pus corpuscles may at 
times contain a red corpuscle. 

Pathogenic Vegetable Parasites. — Of the pathogenic organisms 
which are of especial interest from a clinical standpoint may be 
mentioned the true pus organisms, notably the staphylococci and 
the Streptococcus pyogenes; furthermore, the tubercle bacillus, the 
Actinomyces hominis, the bacillus of glanders, the bacillus of anthrax, 

1 L. Michaelis and A. Wolff, "Die Lymphocyten," Deutsch. med. Woch., 1901, 
vol. xxvii, p. 651. 

2 Bottcher, Virchow's Archiv, 1867, vol. xxxix, p. 512. Bizzozero, loc. cit. 



CHEMISTRY OF EXUDATES 601 

leprosy, tetanus, influenza, and Frankel's pneumococcus, etc. The 
majority of these have already been described. A pathogenic lepto- 
thrix, named by Flexner the L. asteroides, has been found by Cozzo- 
lino 1 in the pus of a retroperitoneal abscess. 

A form of streptothrix has been isolated from the pus of certain 
cases of mycetoma, or Madura foot. 2 

Vincent's 3 fusiform bacilli and spirilla have been encountered in 
the pus of alveolar pyorrhoea, in noma, hospital gangrene, gangrenous 
ulcer of the penis, in bronchiectasis, abscess of the leg, etc. 

In the pus of abscesses in cases of systemic blastomyces infection 
the corresponding organism is found. 

Protozoa, with the exception of the Amoeba coli, have only rarely 
been found. Kiinstler and Pitres 4 observed numerous large spores 
with from ten to twenty crescentic corpuscles in pus taken from the 
pleural cavity of a man, which closely resembled the coccidia of 
mice. Litten a observed cercomonads in the fluid withdrawn from a 
pleural cavity. Trichomonads have been found in empyema in con- 
nection with pulmonary gangrene. 

Most important in this connection is the demonstration of the 
Amoeba coli in the pus, and in cases of liver abscess an examination 
with this end in view should never be neglected. So far as the 
occurrence of amebas in pus is concerned, the observation of Kartulis 
and of Flexner, who demonstrated their presence in an abscess of the 
lower jaw, shows that they should not be looked for in the pus of 
abscesses of the liver or lung only. 

In smears obtained from two cases of oriental boil (tropical ulcer, 
Delhi boil, Aleppo boil) Marzinowsky and Bargow, 6 on the one hand, 
and Wright 7 on the other, found little bodies, measuring from 1 to 
4 fi in diameter and apparently provided with a macronucleus and a 
micronucleus. They are inclined to look upon these as protozoa and 
as parasitic. Marzinowsky and Bragow name the organism Booplasma 
orientale; Wright calls it the Helcosoma tropicum. According to 
Christofers 8 they are identical with the Leishmania-donovani of tropi- 
cal splenomegaly, which latter are known to occur in the skin ulcers 
of kala-azar. 

Vermes. — Of these, the filaria and hydatids are rarely observed 
in this country. Bothriocephalus linguloides has been found in the 
pleural cavity of a Chinese patient. 

1 Zeitsch. f. Hygiene, 1900, vol. xxxiii, p. 36. 

2 Boyce and Adams, Jour. Exper. Med., vol. iii, p. 422. 

3 Annal. de l'lnstitut Pasteur, 1894, vol. viii, p. 129. 

4 Compt.-rend. de la Soc. de biol., 1884, p. 523. 

5 Verhandl. d. Cong. f. inn. Med., 1886, vol. v, p. 417. 

6 Virchow's Archiv, 1904, p. 178. 

7 Jour. Cut. Dis., incl. Syph., New York, June, 1904, and Jour. Med. Research, 
December, 1903. 

8 "Discussion on the Leishman-Donovan Body," Brit. Med. Jour., September 
17, 1904. 



602 TRANSUDATES AND EXUDATES 

Crystals. — As has been stated, crystals of cholesterin are frequently 
found in old pus and in exudates of long standing, but are rarely 
seen in recent exudates. They may be recognized by their charac- 
teristic form and their chemical reactions, as described in the chapter 
on the Feces. Triple phosphates, fatty acid crystals, and hematoidin 
are likewise frequently seen, the presence of the latter, of course, 
indicating a previous admixture of blood. 

The technique to be employed in the examination of pus is as a 
rule simple. Cover-glass preparations or smears on slides are pre- 
pared as in the case of the blood and are then stained according to 
the points that are to be elicited. For routine work the eosinate 
of methylene blue will be found very useful. If the pus corpuscles 
are still fairly fresh, the neutrophilic granules are readily stained; it 
will be noted, however, that very commonly they exhibit a more 
decided red, which is referable to certain degenerative changes which 
cause the granules to assume an affinity for acid dyes as well. Bac- 
teria that may be present are usually well shown. If the pus is 
older and the cells have lost their granules, Pappenheim's pyronin- 
methyl green will be found of value in the study of the mononuclear 
forms. 

Gonorrheal Pus. 

In the very earliest stages of the disease the pus contains large 
numbers of eosinophilic cells besides the common polynuclear neu- 
trophiles. 1 But at the same time and throughout the course of the 
disease mononuclear non-granular elements, with basophilic proto- 
plasm, are also seen. The larger number of the latter are of the type 
of the large mononuclear leukocyte and transition form of Ehrlich, 
but a certain percentage is also represented by the lymphocytes, both 
of the small and large variety. Mast-cells may also occur in gonor- 
rheal pus ; a remarkable case is reported by Neisser, in which the pus 
consisted practically exclusively of such elements. 

The neutrophilic elements in gonorrheal pus commonly present 
evidence of degeneration. In some a loss of granular material has 
manifestly taken place, and it can be demonstrated that in most of 
the cells the granules are no longer absolutely neutrophilic, but have 
become amphophilic — that is, from a neutral mixture they take up 
the neutral dye, but they can also be stained with acid dyes. With 
the triglycerin mixture, for example, they are stained red by the 
eosin. 

As regards the distribution of gonococci in the different cellular 
elements, it is noteworthy that they are principally found in the poly- 

1 Other observers do not mention the early occurrence of eosinophiles in the 
pus. Esserteau states that they are increased from the second to the fourth 
week. 



CHE MIS TR Y OF EX UDA TES 603 

nuclear neutrophils, while they are less commonly seen in the mono- 
nuclear leukocytes and transition forms. In the small lymphocytes 
they are not encountered, and it is uncommon to find them in the 
eosinophilic cells. 

Generally speaking numerous gonococci, eosinophils, and a small 
number of lymphocytes are found in cases of recent gonorrhea, while 
during exacerbations of chronic processes only a few cocci and 
numerous mononuclear elements are encountered. The common 
neutrophilic elements of course control the picture practically at all 
times, so long as there is a discharge. 

The gonococcus (Neisser) (Plate XXI) occurs in the form of 
small oval or coffee-bean-shaped granules, grouped in twos and fours 
resembling a German biscuit; the individual cocci measure about 
1.25 fj. in length by 0.7 /* in diameter. As a rule they are found 
enclosed within pus corpuscles and epithelial cells; but they may 
also occur free in the pus obtained from the urethra, in the vaginal 
discharge, and more rarely in urinary sediments, as in cases of com- 
plicating prostatitis, peri-urethritis, etc. In cover-glass specimens 
account should be taken only of those organisms which are enclosed 
within cellular elements, as these alone may be regarded as charac- 
teristic. To this end a drop of the discharge is spread in a thin 
layer upon a slide or a glover-glass, dried in the air, and fixed by 
passing three or four times through the flame of a Bunsen burner. 
The specimens may then be stained with any one of the basic aniline 
dyes. In my laboratory the eosinate of methylene blue is almost 
exclusively used for this purpose. The organisms are thus colored 
blue, while the granules of eosinophilic leukocytes, which may be 
present at the same time, appear a bright red or a brownish red. 
After five minutes the excess of stain is washed off, the preparations 
are rinsed in water, dried with filter paper, and examined with a 
high power. 

The gonococcus is decolorized by Gram's method and can in this 
manner be distinguished from certain other organisms that may be 
present. Of the four kinds of diplococci which irfey be found in ure- 
thritis besides the gonococcus, only two forms are similarly decolorized, 
and these two are rarely seen. We may conclude that in 95 per 
cent, of all cases Gram's method permits a definite conclusion as to 
the presence or absence of the true organism. Gram's method is 
best employed in the modification suggested by Weinrich: The 
preparations are fixed by drawing through the flame of a Bunsen 
burner and are then stained for from one to two minutes in Frankel's 
carbol-gentian-violet solution (10 parts of a saturated alcoholic 
solution of gentian violet to 90 parts of a 2.5 per cent, solution of 
carbolic acid). Without washing they are placed for one to three 
minutes in Lugol's solution (1 gram of iodine, 2 grams of potassium 
iodide, and 300 c.c. of distilled water), and again without washing 



604 TRANSUDATES AND EXUDATES 

in absolute alcohol, until the alcohol ceases to extract color (about 
one and one-half minutes); they are now washed in water, counter- 
stained with Bismarck brown, washed, dried, and mounted. The 
Bismarck-brown solution is prepared as follows: 3 grams of the dye 
are dissolved in 70 c.c. of hot water; 30 c.c. of 96 per cent, alcohol 
are added; the mixture is well stirred and filtered. 

The organism grows best on blood and hydrocele agar. The sur- 
face colonies are pale, grayish, translucent, and finely granular, with 
finely notched borders. In bouillon and blood serum mixed it forms 
a membrane, while the fluid remains clear. 

When no discharge can be obtained from the urethra, or an exam- 
ination of such discharge is negative, positive results may at times 
still be obtained if some of the gonorrheal threads are examined, 
which may be found floating in the urine. In these the organisms 
can occasionally be demonstrated after months and even years have 
elapsed after primary infection. 

Literature. — Janowski, Arch. f. exper. Pathol., 1895, vol. xxxvi, p. 15. L. 
Michaelis and A. Wolff, "Die Lymphocyten," Deutsch. med. Woch., 1901, vol. 
xxvii, p. 651. A. Pappenheim, Virchow's Archiv, 1901, vol. clxix, p. 72. Neisser, 
Centralbl. f. d. med. Wiss., 1879, vol. xvii, p. 497. J. Plato, "Ueber Gonokok- 
kenfarbung mit Neutralroth," etc., Berlin, klin. Woch., 1899, p. 1085. E. R. 
Owings, "The Infectiousness of Chronic Urethritis," Bull. Johns Hopkins Hosp., 
1897, p. 210. H. H. Young, "Welch Festschrift," Johns Hopkins Press, 1900, 
p. 677. 

Putrid Exudates. 

Putrid exudates are observed following perforation of a gangren- 
ous focus or of a gastric or intestinal ulcer into one of the body 
cavities. At other times they are encountered in cases of neoplasm, 
and at times even without apparent cause. The material obtained 
in such cases has a brown or brownish-green color, and emits an 
odor which in itself indicates the character of the exudate. Micro- 
scopically, cholesterin, hematoidin, and fatty acid crystals, as well 
as degenerating leukocytes, are found. In cases in which aspiration 
of a higher intercostal space reveals the presence of serous fluid, 
while putrid material is obtained at a lower point, the existence of a 
subphrenic abscess should be suspected. In such cases a pure cul- 
ture of the Bacillus coli communis has been obtained. The reaction 
of putrid exudates is usually alkaline, but an acid reaction may be 
obtained in cases of perforation of a gastric ulcer; the Sarcina ven- 
triculi and saccharomyces may then also be found. 

Chylous and Chyloid Exudates. 

Chylous and chyloid exudates have been repeatedly observed. 
They are most frequently met with in the abdominal cavity (one 



CHEMISTRY OF EXUDATES 605 

hundred and four times out of a total number of one hundred and 
fifty-five, which have thus far been reported), less commonly in the 
pleural cavity (forty-nine times), and only rarely in the pericardial sac 
(twice only) (1904). Among the causes which may lead to chylous 
ascites the following are recognized (in the order of their frequency) : 
compression of the thoracic duct or the lymphatic vessels by glandular 
enlargements, neoplasms, etc.; non-tuberculous peritonitis; occlusion 
of the left subclavian; excessive pressure, strain, cough; peritoneal 
carcinoma; filariasis; occlusion of the thoracic duct; occlusion of lymph 
vessels, external pressure; diseases of the liver, syphilis, primary 
disease of the lymph vessels, angioma, calculus of the receptaculum 
chyli, and Hodgkin's disease. Quincke believes that the two forms 
can be etiologically distinguished from one another by means of a 
microscopic examination, as the cloudy appearance in the chyloid 
form is usually referable to the presence of endothelial or epithelioid 
cells undergoing fatty degeneration. Later observations, however, 
have shown that the differentiation of the two forms cannot be made 
upon this basis, as the same anatomical lesion, such as carcinoma or 
tuberculosis, may at times give rise to the formation of a chylous 
exudate, at others to that of the chyloid form, and both, moreover, 
may coexist. An instance of this kind is described by Wilson. 

Senator claimed that the presence of more than traces of sugar is 
strongly suggestive of the chylous nature of the exudate. Possibly 
this observation may be of some value, but only the presence of more 
than 0.2 per cent, is of value. Of greater significance is the fact that 
in chylous fluid the melting point of the fat will depend upon the 
melting point of the fat which was taken in as food, while this is not 
the case in chyloid effusions. The amount of fat, moreover, which 
is present is influenced directly by the amount ingested in the first 
instance. 

Occasionally one can get the distinct odor of the food which has 
been taken, in chylous exudates, while in the chyloid type this would 
hardly be expected. 

Chylous exudates in their general appearance resemble milk, while 
chyloid fluid is more suggestive of pus. The turbidity in both cases 
is usually referable to the presence of innumerable fat globules, 
which are especially abundant in the chylous form. In chyloid 
exudates the origin of the fat from cellular elements is often appar- 
ent at once; but, as has been said, it is impossible to draw definite 
etiological conclusions from that difference. Some chyloid exudates 
contain no fat at all, and Lion has shown that the milky appearance 
in such cases is owing to the presence of a curious albuminous sub- 
stance, belonging to the class of nucleo-albumins. Bernert, on the 
other hand, claims that the substance in question belongs to the 
globulins, and is closely associated with certain lecithins. A similar 
observation is recorded by Micheli and Mattirolo. 



606 TRANSUDATES AND EXUDATES 

Edsall (cited by Wilson) reported an instance of non-fatty pleural 
effusion, the opacity of which was due to altered globulins. 

Chemical analysis of a chylous exudate (pleural) from a case of 
Hodgkin's disease, which Campbell made in my laboratory, showed 
the following result: 

Water . " 90 . 84 per cent. 

Solids 9.15 

Mineral solids . . 0.76 

Organic solids 8 . 39 

Coagulable albumins 4 . 80 

Fats . .3.0 

Sugar 0.59 

The specific gravity was 1.020. 

The cytological formula in such exudates has as jet received but 
little attention. In Campbell's case only a small number of leuko- 
cytes was present and most of these were of the lymphocytic type. 
In Muttermilch's case lymphocytes were said to preponderate; in 
addition there were small numbers of neutrophilic leukocytes, con- 
taining fat granules, together with eosinophilic cells and a very few 
red cells. In the mixed case of Wilson the lymphocytes numbered 
76 per cent., and the large mononuclear cells 22 per cent. 

Literature. — Quincke, loc. cit. Boulengier, Schmidt's Jahrb., 1890, vol. 
ccxxvi, p. 28. Wilson, Amer. Jour. Med. Sci., October, 1905. Boston, Jour. 
Amer. Med. Assoc, February 18, 1905. Micheli and Mattirolo, Wien. klin. Woch., 
1900, No. 3. Muttermilch, Zeit. f. klin. Med., vol. xlvi, p. 123. Shaw, Jour. 
Pathol, and Bacter., vol. vi, 1900. 



Examination of Syphilitic Material. 

Spirochete Pallida. — Through the researches of Schaudinn and 
Hoffmann it has been ascertained that in primary and secondary 
syphilitic lesions a spirochete can be demonstrated which probably 
represents the cause of the disease. Their results have been abun- 
dantly verified both abroad and in the United States. The organism 
has been demonstrated in the scrapings obtained from chancres, 
incised papules and condylomata, and in smears from mucous patches 
and the aspirated juice of the inguinal glands. Schaudinn and Hoff- 
mann could further demonstrate the organism in the blood obtained 
by puncture of the spleen in a recent case of syphilis on the day pre- 
ceding the eruption. Levaditi found it in the vesicular contents of 
pemphigus syphiliticus. Buschke and Fischer, Babes and Panea, 
and Levaditi found the spirochete in the internal organs of children 
which had died of congenital syphilis, as also in the blood, and 
Metschnikoff could demonstrate it in the lesions of artificial syphilis 
in the ape. 

The Spirochete pallida derives its name from its low refractive 
power and the difficulty with which it takes up aniline dyes (this 



S pi roch setae 



CHEMISTB Y OF EXUBA TES 607 

especially in contradistinction to the Spirochete refringens). It is 
a very delicate structure, usually presenting 10 to 40 deep spiral incur- 
vations with the larger specimens, or only 2 to 4 in the smaller ones. 
The length varies from 4 to 10 (jl with 7 ft as an average; the width 
does not exceed 0.5 ft. In the wet preparation it*may be observed 
that its movements occur in an oscillatory manner about the longi- 
tudinal axis, and that, in contradistinction to the spirilla, the move- 
ments of the spirochete are winding, bending, and whipping, while in 
the spirilla the longitudinal axis remains rigid. Schaudinn also 
demonstrated the existence of a flagellum at each end, while the other 
spirochetas have an undulating membrane. (See Plate XXII and 
Fig. 171). 




Fig. 171. — Spirochsete pallida. 

Staining Methods. — Excellent results are obtained with Goldhorn's 
stain (which see). To this end the smears, on slides or covers, are 
covered with the dye for three or four seconds, when the excess is 
drained off. The specimens are then introduced slowly into clean 
water with the film side down, permitting in this manner an interaction 
between the film of adhering dye and the water. The slide is held in 
this slanting position for another four or five seconds and is next shaken 
in the water so as to wash off the excess of the dye. The pallida 
appears of a violet color, which may be changed to bluish black by 
flooding the preparation for fifteen to twenty seconds with Gram's 
iodine solution, washing and drying as usual. The examination is 
conducted with a ^ or T 1 g- immersion lens. 

The material should in all cases be obtained by curettage, this 
being carried so far until a small amount of serum and blood appears, 



608 TRANSUDATES AND EXUDATES 

and preferably at the edge of the lesion. The serous fluid is then 
spread upon slides or covers in the usual manner. The organisms 
are most numerous in moist papules and chancres (when the curettage 
is carried out at the edge of the lesion). In roseolar scrapings the 
search is frequently disappointing. 

Giemsa's method also furnishes excellent results. I have recom- 
mended a dilute alcoholic solution of Victoria blue. Keidel finds 
that steaming the specimens with this solution for a minute or two 
furnishes good results. 

Literature. — Schaudinn and Hoffmann, Arbeiten aus d. kais. Gesundheits- 
amte, 1905, vol. xxii, p. 527; Deutsch. med. Woch., 1905, No. 18, p. 711 ; Berlin, 
klin. Woch., 1905, May 29, p. 673. Metschnikoff and Roux, Le bulletin med., 
May 17, 1905, p. 441. Levaditi, La semaine med., May 24, 1905, p. 247. Hoff- 
mann, Berlin, klin. Woch., 1905, No. 22, p. 673. Fanoni, Med. News, October, 
1905. Babes and Panea, Berlin, klin. Woch., 1905, No. 28, p. 865. Mulzer, 
ibid., No. 36, p. 1144. Goldhorn, Jour. Exper. Med., 1906, vol. viii, No. 3. 



CHAPTER IX. 

THE CEREBROSPINAL FLUID. 

According to our present knowledge, the cerebrospinal fluid is 
secreted by the choroid plexuses into the lateral ventricles. Passing 
through the foramina of Monro, the third ventricle, and the aque- 
duct of Sylvius, on the one hand, it reaches the fourth ventricle and 
enters the cystern-like subarachnoid spaces at the base of the brain, 
through the foramen of Magendie and the lateral clefts of the fourth 
ventricle. On the other hand, a certain portion of the fluid reaches 
the same destination directly through the cleft in the descending horn 
of each lateral ventricle. The larger portion of the fluid then passes 
upward through the subarachnoid spaces along the convexity of the 
brain to the Pacchionian granulations, while the smaller portion 
enters the vertebral canal through the subarachnoid spaces of the 
spinal arachnoid membrane. 

Within recent years puncture of the vertebral canal has been 
frequently resorted to, both for therapeutic and diagnostic purposes. 
The practical value of this method of diagnosis is now beyond ques- 
tion, and it is to be hoped that ere long physicians will resort to 
spinal puncture in obscure cases of cerebrospinal disease with as 
little hesitancy as puncture of the thoracic and abdominal cavities is 
now practised. 1 

The operative method to be employed is the following : With the 
patient placed upon his left side — some observers prefer the sitting 
posture — and the body bent well forward, a long aspirating needle 
is introduced upon a level with the lower third of the third or fourth 
lumbar spinous process, and about 1 cm. to the side of the median 
line, the needle being directed slightly upward and inward. The 
depth to which it is necessary to puncture will, of course, vary with 
the age of the patient. In a child two years of age the vertebral 
canal may be reached at a depth of 2 cm., while in the adult it is 
necessary to insert the needle for a distance of from 4 to 8 cm. As 
soon as the subarachnoid space is reached cerebrospinal fluid will 
flow from the needle. Aspiration should always be avoided. 

Some writers have advised that the operation be performed under 

1 H. Quincke, Verhandl. d. X Cong. f. inn. Med., 1891. A. Hand, " A Critical 

Summary of the Literature on the Diagnostic and Therapeutic Value of Lumbar 

Puncture," Amer. Jour. Med. Sci., 1900, vol. cxx, p.463. A. Stadelmann, "Klin- 

ische Erfahrungen mit d. Lumbalpunction," Deutsch. med. Woch., 1897, p. 745. 

39 



010 THE CEREBROSPINAL FLUID 

narcosis; and without doubt this may be necessary at times, particu- 
larly when contracture of the dorsal muscles exists. In the majority 
of cases, however, it is not necessary and local anesthesia will suffice. 

Amount. — So far as I have been able to ascertain, no observations 
have been made regarding the amount of fluid which may be obtained 
by puncture in normal individuals. In all probability, however, this 
is small. Under pathological conditions the amount may vary from 
a few drops to 100 c.c, and even more. In general terms it may 
be stated that the amount is directly proportionate to the degree of 
intracranial pressure. Exceptions, however, are frequent. Small 
amounts of cerebrospinal fluid or none at all may thus be obtained 
when, owing to the formation of a thick exudate or the existence of 
a cerebral tumor, communication between the basilar subarachnoid 
spaces of the brain and those of the spinal cord has been interrupted. 
Whenever, then, symptoms of intracranial pressure exist, while no 
fluid or minimal amounts only can be obtained by puncture, the 
conclusion will usually be justifiable that we are dealing with a 
purulent meningitis or with a tumor of the brain, and more especially 
of the cerebellum. It should be remembered, however, that the 
same result may be obtained in cases of obliteration of the aqueduct 
of Sylvius, or when sclerotic processes involve the foramen of 
Magendie, which is occasionally observed in certain forms of hydro- 
cephalus. Adhesions of the pia mater to the arachnoid and the 
dura mater may, by interfering with the flow of cerebrospinal fluid, 
also lead to the formation of hydrocephalus, but in these cases a 
tumor can usually be excluded, as the changes in question always 
develop as sequels to a meningitis. A serous or tuberculous menin- 
gitis, as well as acute hydrocephalus and tetanus, can, however, 
always be excluded when only minimal amounts of fluid are obtained 
by puncture. The largest amounts, on the other hand, are seen in 
cases of serous meningitis, tuberculous meningitis, and cerebral tumors, 
which do not interfere with the circulation of the cerebrospinal 
fluid. In the epidemic type of meningitis 70 to 80 c.c. can usually 
be obtained very readily. In epilepsy Pellagrini usually obtained 
amounts varying between 10 and 15 c.c. 1 Donath gives rather higher 
figures, up to 60 c.c, and in a tabes case 85 c.c. 

Appearance. — Normal cerebrospinal fluid, as well as that obtained 
in cases of serous meningitis, tuberculous meningitis, hydrocephalus, 
and tumors of the brain, is perfectly clear, and as a rule colorless 
unless a small bloodvessel has been punctured, when the fluid may 
present a slightly reddish tinge. More or less pronounced yellow 
shades are, however, at times observed. Important from the stand- 
point of diagnosis is the fact that in cases of hemorrhage into the 
ventricles pure blood is obtained, while such a result is, of course, a 

1 La Riforma med., 1901, Ann. 17, vol. ii, p. 638. 



THE CEREBROSPINAL FLUID 611 

mechanical impossibility in cases of epidural hematoma. In subdural 
hematoma, on the other hand, blood may also find its way into the 
subarachnoid space, but the amount is always small, and cannot be 
compared with that seen in cases of ventricular hemorrhage. When- 
ever, then, as in traumatic cases with severe cerebral symptoms, the 
surgeon is confronted with the question whether or not to trephine, 
puncture of the subarachnoid space may furnish much valuable 
information. If in such cases no blood at all is found, it may be 
inferred that an epidural hematoma or a subdural hematoma of 
slight extent only exists; an operation may then be performed. If, 
however, pure blood is encountered, it would be justifiable to assume 
the existence of extensive injury to the brain substance proper, or, 
in cases in which the history is obscure, an intracerebral hemorrhage 
with rupture into the ventricles. In such cases the idea of an oper- 
ation would, of course, be entertained only under exceptional con- 
ditions. If, further, the fluid is only tinged with blood, a subdural 
hematoma probably exists, and an operation should be advised. 
Accidental hemorrhage, viz., hemorrhage referable to the puncture 
itself, can be readily recognized, as the first few drops only are then 
tinged with blood, or the blood appears only after the flow has been 
definitely established; the amount, moreover, is insignificant. 

Cloudy fluid is obtained in all cases of purulent meningitis unless 
the disease is limited to a very small area. In the epidemic type, 
however, it may be quite clear, or but slightly cloudy. Cases of ab- 
scess of the brain or sinus thrombosis occur again and again in which 
the question as to the advisability of operative interference is largely 
dependent upon the presence or absence of a complicating purulent 
meningitis. In certain instances a satisfactory conclusion may, of 
course, be reached without puncture; but in many others this is 
impossible, and Lichtheim's dictum, that an operation should never 
be undertaken in such cases unless the integrity of the meninges has 
been established by spinal puncture, should be borne in mind. 

The degree of cloudiness naturally varies in different cases, and 
while in some instances the character of the fluid is seropurulent, 
pure, creamy pus may be found in others. Generally speaking, a 
cloudy fluid indicates the existence of an acute inflammatory process 
or an exacerbation of a chronic process. 

Important, furthermore, is the fact that the fluid in non-inflam- 
matory diseases of the brain, such as tumor or abscess, rarely under- 
goes coagulation, while this is the rule in all inflammatory diseases. 
In tuberculous meningitis the coagula are very delicate, and may be 
well compared with spider-webs extending throughout the fluid, while 
in purulent meningitis the coagula are somewhat firmer. 

Specific Gravity. — The specific gravity of cerebrospinal fluid 
normally varies between 1.005 and 1.007, corresponding to the pres- 
ence of from 10 to 15 pro mille of solids. Under pathological con- 



612 THE CEREBROSPINAL FLUID 

ditions variations from 1.003 to 1.012 may be observed, the specific 
gravity, generally speaking, being higher in the inflammatory than 
in the non-inflammatory diseases of the brain. From a diagnostic 
standpoint, however, the determination of the specific gravity is of 
little value, as numerous exceptions occur to the above rule. 
The reaction is always alkaline. 



CHEMICAL COMPOSITION OF CEREBROSPINAL FLUID. 

An idea of the chemical composition of the cerebrospinal fluid may 
be formed from the following analyses, taken from Gautier and 
Zdarek : 

Per cent. 

Water 987.00 

Albumin 1.10 

Fat 0.09 

Cholesterin 0.21 

Alcoholic and aqueous extract, minus salts "I „ ., 

Sodium lactate . j * " " " " " ^<a 

Chlorides 6.14 

Earthy phosphates 0.10 

Sulphates . 20 

Zdarek's Analysis. 

Water .. 989.54 

Solids 10.45 

Organic solids . 2.09 

Mineral ash . 8 . 35 

Albumins 0.76 

Ethereal residue . 35 

Aqueous residue 8 . 22 

Sulphuric acid (S0 3 ) . 04 

Chlorine 4.24 

Carbon dioxide . 49 

Potassium oxide 0.16 

Sodium oxide 4 . 29 

Mineral ash, insoluble in water 0.16 

Glucose 0.10 

In addition, urea is at times found, as also a substance which 
reduces Fehling's solution and gives rise to a brown color when 
boiled with caustic potash, but which neither undergoes fermentation 
nor forms an osazone when treated with phenylhydrazin. The sub- 
stance in question is generally regarded as pyrocatechin. Its amount 
varies between 0.002 and 0.116 per cent. According to C. Ber- 
nard, glucose may also be present, but it is questionable whether 
this is the case under normal conditions (see below). Nawratzki 
discovered a reducing substance in his cases, which was demon- 
strated to be glucose; his subjects, however, were unfortunately not 
normal, but general paretics with fever. Pyrocatechin was absent. 
Zdarek 1 reports a recent case of anterior meningocele in an otherwise 

1 Zeit. f. phys. Chem., 1902, vol. xxxv, p. 202 



CHEMICAL COMPOSITION OF CEREBROSPINAL FLUID 613 

normal individual in which the fluid reduced Fehling's solution and 
gave a glucosazone with phenylhydrazin. The substance in question 
was dextrorotatory, the amount equalling 0.1 per cent, of glucose. 

Lichtheim claims to have found glucose — by means of the phenyl- 
hydrazin test — in all cases of tumor which he examined. In cases 
of tuberculous meningitis, on the other hand, a positive result was 
only exceptionally obtained. Quincke also reports that he was able 
to demonstrate the presence of sugar whenever the liquid obtained 
was sufficient in amount for the necessary tests. Unfortunately, 
however, he does not detail his cases. Concetti found no sugar in 
hydrocephalic fluid. 

The experience of other observers does not agree with that of 
Lichtheim and Quincke; and Fiirbringer, 1 who has thus far reported 
the largest number of spinal punctures, found sugar in only 2 cases 
of diabetes associated with tuberculosis. 

So far as the albuminous bodies are concerned which may be found 
in the cerebrospinal fluid, serum albumin is said. to be present only 
under exceptional conditions, while normally a mixture of globulin 
and albumoses is found. The question whether or not mucin may 
also be present is still undecided. 2 

Under pathological conditions the amount of albumin may vary 
considerably, and is of diagnostic importance. According to the 
majority of observers, the figure given in the above analysis is too 
high, and it is doubtful whether 1 pro mille may be regarded as 
normal. The lowest values have been obtained in cases of chronic 
hydrocephalus (traces only), meningitis serosa (0.5 to 0.75 pro mille), 
and tumors of the brain (traces to 0.8 pro mille); while the largest 
amounts have been found in chronic hydrocephalus the result of 
hyperemia (1 to 7 pro mille), and in tuberculous meningitis (1 to 3 
pro mille). Nawratzki in recent examinations found amounts vary- 
ing between 0.047 and 0.170 per cent., but the subjects of his investi- 
gation had fever at the time. Mott and Halliburton 3 found three 
times the normal amount of albumin in paralytics, as also some 
nucleo-albumin, which does not occur in health. The latter they sup- 
pose to come from broken-down Nissl bodies. 

Cholin. — According to Gumprecht, the normal cerebrospinal fluid 
also contains traces of cholin. Donath obtained positive results 
(using 10 to 20 c.c.) in 15 cases of genuine epilepsy out of 18, three 
times in 3 cases of Jacksonian epilepsy, once in a case of syphilitic 
epilepsy, twice in 3 cases of dementia paralytica, once in 2 cases of 
taboparalysis, ten times in 15 cases of tabes dorsalis, three times in 3 

1 Verhandl. d. XV Cong. f. inn. Med., 1901. 

2 Stadelmann, Mitth. a.d. Grenzgebieten d. Med. u. Chir., vol. ii. Comba, Clin, 
med., 1899 (cited in Arch. d. med. d. enfants, 1900). Lenhartz, Verhandl. d. 
XIV Cong. f. inn. Med., 1900. 

3 The Lancet, April, 1901. 



614 THE CEREBROSPINAL FLUID 

cases of cerebral syphilis, twice in 2 cases of cerebral abscess, once in 
a case of encephalomalacia, once in a case of spina bifida, once in a 
case of compression myelitis, once in a case of alcoholic polyneuritis, 
once in 3 cases of neurasthenia, and once in 3 cases of hystero- 
epilepsy. Negative results were obtained in 2 cases of hysteria and in 
multiple cerebrospinal sclerosis. Quantitative estimations were made 
in 10 cases; the amounts varied between 0.021 and 0.046 per cent. 

Method. — According to Donath, 1 the cerebrospinal fluid (10 to 
30 c.c.) is collected in test-tubes, feebly acidified with dilute hydro- 
chloric acid, and evaporated to dryness on the water bath. The 
dark (orange yellow to dark brown) residue is extracted with absolute 
alcohol (99 per cent, is not sufficient), and the filtered solution treated 
with a solution of platinum chloride in absolute alcohol. On standing 
the chloroplatinate of cholin separates out. This can be identified 
by its ready solubility in cold water (as contrasted with the very 
slight solubility of potassium and ammonium platinochloride) and 
its very characteristic crystals. These are usually serrated and lan- 
ceolated or leaf-wreath or rosette shaped, the latter with three or 
four leaves. Occasionally they are radiate needles, or needles arranged 
in sheaves (obliquely cut prisms) or hexagonal or rhombic platelets. 
They are commonly tinged yellow, but if very thin (especially the 
needles) they appear colorless. The crystals are best obtained by 
allowing a few drops of their aqueous solution to evaporate on a slide. 

The alkaline platihochlorides appear as octohedra or tetrahedra, 
which may have blunt angles; but according to Donath they are never 
seen with the method as above outlined (using absolute alcohol — 
alcohol dehydrated with anhydrous copper sulphate and kept over 
this). 

Another delicate reagent for cholin in aqueous solution is phos- 
photungstic acid. In dilute solutions a white precipitate will form 
which appears under the microscope as composed of small hexagonal 
plates or rhomboids. As chloride of potassium and ammonium will 
also give a precipitate with phosphotungstic acid, the extract in 
absolute alcohol (see above) should be filtered, the alcohol evapo- 
rated, and the residue dissolved in water. 

The physiological test for cholin, viz., fall in blood pressure follow- 
ing its intravenous injection in aqueous solution, is usually unnecessary. 

Coriat 2 found cholin invariably present in general paresis, also in 
1 case of central neuritis, in 2 alcoholic cases with polyneuritis, in 1 
of senile dementia, in 1 of senile dementia associated with a tumor 
in the corpus callosum, in 1 of traumatic organic dementia, also 
associated with a tumor of the corpus callosum. The largest amounts 
were found in paresis. Lecithin was found twice by Donath, once in 
a tabes case and once in Jacksonian epilepsy. 

1 Med. News, January 21, 1905. 

2 Amer. Jour, Insanity, 1904, No. 4. 



MICE SCO PIC EXAMINA T10N 615 



MICROSCOPIC EXAMINATION. 

Cytology. — Normal cerebrospinal fluid contains either no morpho- 
logical elements at all or only a small number of lymphocytes (three to 
eight to a field, with a medium power). Deviations from this 
normal condition, as has been first shown by Widal, Ravaut, Sicard, 
and others, may be of marked diagnostic value. 

Aside from tuberculous meningitis in which lymphocytosis is practic- 
ally constant an increased number of lymphocytes has been observed in 
syphilitic lesions of the central nervous system (general paresis, tabes, 
cerebrospinal syphilis, syphilitic hemiplegia), in certain cases of 
herpes zoster, sciatica, and parotitis. Of these the syphilitic cases are 
most important, but it is to be noted that the increase may be inter- 
mittent and paroxysmal. As a rule it is well marked. Lymphocytosis 
also occurs in lead intoxication and in saturnine encephalopathy it 
may be quite intense. The same has been noted in African sleeping 
sickness. Negative results have been obtained in poliomyelitis, 
syringomyelia, the hemiplegia of old age, polyneuritis, functional 
neuroses, compression myelitis, cerebral tumors, and epilepsy. 

According to Niedner, lymphocytosis is quite constant in syphilitic 
hemiplegia, while it is inconstant in tabes. Of 9 cases reported by 
Niedner and Mamlock, 1 lymphocytosis occurred in 5. In general 
paresis lymphocytosis is very common. 

In the epidemic form of cerebrospinal meningitis the predominat- 
ing cell is the polynuclear neutrophile, excepting in chronic cases 
where lymphocytes may prevail. This cell also enters into the 
foreground as recovery occurs. 

Donath summarizes his results in 98 cases as follows: In acute 
and purulent meningitis polynuclear leukocytes prevail; in chronic 
or less intense processes, especially in tuberculous meningitis, lymph- 
ocytes predominate. In the differential diagnosis of syphilitic men- 
ingitis, the early stages of tabes and of general paresis, from neurotic 
conditions and other malignant processes, lymphocytosis points to 
the first group. In tetanus a large number of polynuclear neutro- 
phils may also occur. 

While in cerebrospinal meningitis referable to the Diplococcus 
pneumoniae polynuclear leukocytosis is probably the rule, exceptions 
occur. Goggia 2 thus reports a fatal case in which daily examina- 
tions showed a predominance of the small mononuclear elements 
throughout the course of the disease. 

In connection with cerebral hemorrhage (especially hemorrhage 
into the ventricles) Sabrazes and Muratet 3 have described the occur- 

1 Zeit. f. klin. Med., 1904, Heft 1 and 2. 

2 Gaz. d. Osped. e. d. clin., 1905, No. 13. 

3 Soc. d. biol., 1903, pp. 1226 and 1435. 



616 THE CEREBROSPINAL FLUID 

rence of large, round, oval, or polyhedral cells, either singly or in 
plaques, provided each with a single oval nucleus containing several 
nucleoli. These cells commonly contain red blood corpuscles, often 
in large numbers, as also crystals and amorphous particles of hema- 
toidin, leukocytic nuclear debris and vacuoles. These cells are macro- 
phages, derived undoubtedly from the endothelial lining of the 
subarachnoid spaces. Besides, granular structures may be met with 
which may contain globules of fat, nuclear debris, globules of myelin, 
red cells, and blood pigment. What these latter cells are is not 
known. Sabrazes inclines to view them as neuroglia cells. 

The technique employed in the cytological study of the cerebro- 
spinal fluid is the same as in the case of pleural exudates. 

Bacteriology. — Very important from a diagnostic standpoint is the 
fact that pathogenic microorganisms may be found. Lichtheim, 
Furbringer, Freyhan, Dennig, Frankel, and many others since, were 
thus able to demonstrate .the presence of tubercle bacilli in a fairly 
large number of cases of tuberculous meningitis. Some observers, it 
is true, have been less fortunate, but the fact that Furbringer found 
tubercle bacilli in 30 cases out of 37 is certainly significant. Schwarz 
states that he obtained positive results in 16 out of 22 cases; Slawyk 
and Manicatide found bacilli in all of 19 cases (sixteen times by 
direct microscopic examination and three times by the animal 
experiment) and Koplik found them in 13 out of 14 cases, using 
centrifugalized material. In order to examine for tubercle bacilli, 
the fluid should be placed on ice for from six to twenty- four hours, 
until a slight coagulum has formed, when the fine, spider-web-like 
threads of fibrin are transferred to a cover-slip, spread in as thin a 
layer as possible, and stained as described in the chapter on the 
Sputum. If a centrifugal machine is available, the examination 
may, of course, be made at once; the chances of finding the bacilli 
are then also much greater. In every case a large number of speci- 
mens should be prepared before the search is abandoned. Only a 
positive result, however, is of value, and in doubtful cases recourse 
should be had to the animal experiment. 

In the diagnosis of epidemic cerebrospinal meningitis lumbar 
puncture is of signal value, as the Diplococcus meningitidis intracellu- 
lars (meningococcus) of Weichselbaum-Jager can be demonstrated in a 
large percentage of cases. Councilman thus states that during a recent 
epidemic of the disease in Boston lumbar puncture was performed 
in 55 cases, and that in the fluid obtained the diplococci were found 
on microscopic examination or in culture in 38 cases. The organism 
was present in all the acute cases, but rarely found in those which 
ran a more chronic course. The average time from the onset of the 
disease before spinal puncture was made was seven days in the posi- 
tive cases and seventeen days in the negative cases. The longest 
time after the onset in which a positive result was obtained was twenty- 



MICROSCOPIC EXAMINATION 



617 



nine days. Similar results have also been reached by other observers. 
Koplik thus found the organism within the first twenty-four hours 
after the onset of the disease and as late as the fifteenth week. In 
chronic cases, however, as Councilman also found, it may escape 
detection, especially in those of the posterior basic type. 

The organism in question is a diplococcus, each half being of about 
the same size as the ordinary pathogenic micrococci (Fig. 172). It 
is readily stained with the usual dyes, and decolorized by Gram's 
method. Short chains of from four to six may at times be seen, 
as also tetrads and peculiarly swollen forms which are much larger 
than the usual forms. Cultivation is difficult and the organism 
quickly dies out. It grows best upon Loffler's blood-serum mixture, 
forming round, whitish, shining, viscid-looking colonies, with smooth, 
sharply defined outlines, which may attain a diameter of from 1 to 
lj mm. in twenty-four hours. Their cultivation upon plain agar, 





Fig. 172. — Diplococcus meningitidis intracellularis. 



glycerin agar, and in bouillon is less reliable. I have obtained excel- 
lent results by placing a few c.c. of the cerebrospinal fluid in blood- 
serum tubes and found that the organisms multiplied far more actively 
in the fluid over the medium than in any other way. 

In order to obtain the best results, it is necessary to use large 
amounts of the exudate, and to make a number of cultures, as many 
of the organisms are usually dead, or at least will not grow. In 
ordinary cover-slip preparations they are often numerous, and are 
found enclosed in the polynuclear leukocytes. Their number then 
varies considerably. On the one hand, only one or two may be 
present in a cell, while in others they may be so closely packed as to 
obscure the nucleus. During the past winter I examined a specimen 
in which the organism was present in groups composed of hundreds, 
but this is rare. 



618 THE CEREBROSPINAL FLUID 

Mixed infections are not uncommon in epidemic cerebrospinal 
meningitis. Councilman thus found the pneumococcus in 7 cases 
and Friedlander's bacillus in 1. Terminal infections with staphy- 
lococci and streptococci also occur. 

In other forms of purulent meningitis a large variety of organisms 
has been found. Wolf gives the following figures, resulting from an 
analysis of 174 cases, in which epidemic cerebrospinal meningitis is, 
however, included: in 44.23 per cent, the pneumococcus was found; 
in 34.48 per cent, the Diplococcus meningitidis intracellularis ; in 
3.45 per cent, staphylococci; in 8.03 per cent, streptococci; in 1.13 
per cent, the bacillus of Friedlander; in 2.87 per cent, the Bacillus 
typhosus; in 1.72 per cent, the bacillus of Neumann-Schaffer, and 
in 2.87 per cent, the Bacillus coli communis, the Bacillus pyogenes 
fcetidus, the Bacillus aerogenes meningitidis, and the Bacillus mallei; 
while no bacteria were found in 1.15 per cent, of the cases. In 
2 cases Pfeiffer's influenza bacillus has also been encountered in the 
cerebrospinal fluid during life. 

In the African sleeping sickness trypano somes are commonly 
found in the cerebrospinal fluid, obtained by lumbar puncture. 
Castellani obtained the organism in 20 cases of 34, and Bruce found 
it in all of 38 cases (see Blood). The results of these earlier observers 
have been abundantly confirmed. In many cases, however, the para- 
sites never find their way into the cerebrospinal fluid. They are 
more frequently found toward the termination of the disease. Large 
numbers are rare, but if they do occur there is usually an access of 
temperature. When present, the leukocytes are apt to be increased. 
There is no relation between the number present in the blood and in 
the spinal fluid. 

Toxicity. — While normal cerebrospinal fluid possesses distinct 
toxic properties, it has been found that in disease the toxicity may 
be markedly increased. Bellisari has thus shown that the fluid of 
individuals suffering from general paresis is more toxic than that 
of normal individuals, and that this toxicity is at its maximum after 
an epileptic seizure. Pellegrini further could demonstrate that the 
cerebrospinal fluid of epileptics is markedly toxic, and that that 
obtained immediately after a convulsion has a toxic and convulsive 
power much greater than that obtained at periods far removed 
from paroxysms. Similar results have been obtained by Dide and 
Laquepee. 

Literature. — W. T. Councilman, "Cerebrospinal Meningitis," Johns Hopkins 
Hospital Bull., 1898, p. 27; and Phila. Med. Jour., 1898, p. 937. W. T. Council- 
man, F. B. Mallory, and J. H. Wright, "Epidemic Cerebrospinal Meningitis," 
Amer. Jour. Med Sci., 1898, p. 252. W. Osier, "The Cavendish Lecture on the 
^Etiology and Diagnosis of Cerebrospinal Fever," Phila. Med. Jour., 1899, p. 26. 
E. Stadelmann, "Meningitis Cerebrospinalis," Zeit. f. klin. Med., vol. xxxviii, p. 
46. R. Neurath, Centralbl. f. d. Grenzgebiete d. Med. u. Chir., 1897, vol. i. 
J. Langer, Jahrb. f. Kinderheilk., 1901, vol. iii, p. 91. Pellegrini, Riform. med., 
1901, No. 55. Dide and Laquepee, Soc. d. neurol. de Paris, April, 18, 1901. 



CHAPTER X. 
THE EXAMINATION OF CYSTIC CONTENTS. 
CYSTS OF THE OVARIES AND THEIR APPENDAGES. 

The material obtained from cysts of the ovaries or their appen- 
dages varies greatly in character. On the one hand, it may be 
fluid, clear, of low specific gravity, and contain little albumin; while, 
on the other, it may be dense, viscid, and of colloid appearance. 
The specific gravity varies between 1.018 and 1.024, owing to the 
presence of a large amount of albumin. 

In addition to smaller amounts of serum albumin and serum 
globulin the fluid of ovarian cysts contains a considerable quantity 
of another albuminous substance, which is termed metalbumin 
(Scherer) or pseudomucin (Hammarsten). Like Hammarsten's 
mucoid of transudates, it cannot be directly precipitated with acetic 
acid, but must be isolated as follows: The fluid in question is freed 
from coagulable albumins by boiling after acidifying with acetic acid ; 
the filtrate is precipitated with alcohol, the precipitate dissolved in 
water, dialyzed, and then treated with acetic acid, when the pseudo- 
mucin separates out. The substance contains about 30 per cent, of 
glucosamin. 

Paramucin is another albuminous substance which is found in 
colloid cysts and belongs to the mucinoid bodies. Like the true 
mucins and the body which occurs in exudates the paramucin is 
also precipitated by dilute acetic acid. According to Mitjukoff, it 
contains at least 12.5 per cent, of a reducing substance. 1 

Test for Pseudomucin.— The fluid is mixed with three times its 
volume of alcohol and set aside for twenty-four hours, when it is 
filtered and the precipitate suspended in water. This is again 
filtered and the filtrate tested in the following manner: (1) A few 
cubic centimeters are boiled, when in the presence of metalbumin 
the liquid will become cloudy, without the formation of a precipitate. 
(2) With acetic acid no precipitate is obtained. (3) Upon the appli- 
cation of the acetic acid and potassium ferrocyanide test the liquid 

1 Literature dealing with pseudomucin and paramucin: Pseudomucin: Ham- 
marsten, Zeit. f. phys. Chem., 1882, vol. vi, p. 194. Pfannenstiel, Arch. f. Gynak., 
1890. Zangerle, Munch, med. Woch., 1900. Paramucin: Mitjukoff, Arch. f. 
Gynak., 1895. Panzer, Zeit. f. phys. Chem., 1899, vol. xxviii. Leathes, Arch, 
f. exper. Path. u. Pharmak., 1899, vol. xliii. 



620 



THE EXAMINATION OF CYSTIC CONTENTS 



becomes thick and assumes a yellowish color. (4) When boiled with 
Millon's reagent a few cubic centimeters of the filtrate will yield a 
bluish-red color, while the addition of concentrated sulphuric acid, 
without boiling, gives rise to a violet color. 

The color of cystic fluids may vary from a light straw to a reddish 
brown, or even a chocolate; the latter color may be observed when 
hemorrhage has taken place into the cyst. 

Of morphological elements, ovarian cysts contain red blood cor- 
puscles, leukocytes, and at times fatty granules in large numbers, 
crystals of cholesterin, hematoidin, and fatty acids. Most im- 
portant, however, from a diagnostic standpoint is the presence of 
cylindrical or prismatic, ciliated epithelial cells, derived from the 




Fig. 173. — Contents of an ovarian cyst: a, squamous epithelial cells; b, ciliated epithelial 
cells: c, columnar epithelial cells; d, various forms of epithelial cells; e, fatty squamous 
epithelial cells; /.colloid bodies; g, cholesterin crystals. (Eye-piece III, obj. 8 A, Reichert.) 
(v. Jaksch.) 

internal lining of the cyst, in the presence of which the diagnosis 
may be definitely made (Fig. 173). At times such cells cannot be 
demonstrated, as they may have undergone fatty degeneration; 
moreover, if the epithelium lining the cyst is squamous in character, 
it may be difficult, if not impossible, to arrive at a satisfactory con- 
clusion from an examination of the morphological elements alone. 
Colloid concretions, which may vary in size from several micromil- 
limeters to 0.1 mm., are occasionally observed, and more particu- 
larly in colloid cysts. They may be recognized by their irregular 
form, homogeneous appearance, slightly yellow color, and delicate 
outlines. 

In dermoid cysts, epidermal cells and occasionally hairs are ob- 
served. 



HYDATID CYSTS 621 

The differential diagnosis of ovarian, parovarian, and fibrocystic 
(uterine) cysts cannot always be made from the character of the fluid 
withdrawn by puncture, but at times it is possible. The most im- 
portant points of difference are here given: (1) The fluid in ovarian 
cystomas is usually more or less viscid, and often contains non- 
nucleated granular corpuscles of about the size of leukocytes, the 
granules of which do not dissolve in acetic acid nor disappear when 
treated with ether. In all probability they are free nuclei; in the 
United States they are often called Drysdale's corpuscles. (2) In 
parovarian cysts the fluid is thin, watery, of low specific gravity 
(under 1.010), and contains very few morphological elements. 
Cylindrical epithelium is very rarely found during life in the fluid 
withdrawn by aspiration from either ovarian or parovarian cysts. 
(3) The fluid from fibrocystic tumors of the uterus is thin, watery, 
and coagulates spontaneously, while that from ovarian and paro- 
varian cysts never coagulates spontaneously unless blood is present. 
Fibrocystic tumors of the uterus have no epithelial lining. 

Of special interest are those cases of ovarian cysts in which in 
the course of typhoid fever infection of the cystic contents occurs 
with the corresponding organism. 1 

HYDATID CYSTS. 

The normal fluid in hydatid cysts is clear like water, neutral (some- 
times faintly acid or alkaline), of a specific gravity of 1.000 to 1.015, 
and rich in sodium chloride. By transmitted light it is faintly opales- 
cent. It contains no albumin or only a trace of it. Succinic acid or 
sugar may be present in small amount. Sodium chloride may be 
recognized by evaporating a drop of the liquid on a slide, when the 
characteristic crystals of the salt will be found. Succinic acid may 
be demonstrated by acidifying a small amount of the fluid with hydro- 
chloric acid, and evaporating to dryness. The residue is extracted 
with ether and the ether evaporated; the aqueous solution of the 
second residue, in the presence of succinic acid, will yield a rust- 
colored, gelatinous precipitate when treated with a few drops of a 
solution of ferric chloride. A sediment, if present, is composed chiefly 
of scolices, debris of parenchyma, calcareous particles, and hooklets. 
Hematoidin crystals may be found if blood has entered the cyst. 
Where tapping or exploratory puncture has been employed, albumin 
may afterward be found in greater quantity, as also in degenerating 
and suppurating cases. With the death of the hydatid, changes of a 
degenerative nature take place, the fluid altering greatly in character. 
It becomes more turbid, fatty globules may be found with granular 

1 M. J. Lewis and R. G. Le Conte, Amer. Jour. Med. Sci., 1902, vol. cxxiv, p. 
590. 



622 THE EXAMINATION OF CYSTIC CONTENTS 

cells, and typical crystals of cholesterin. The contents may become 
of putty-like consistence and greasy, containing the remains of the 
gelatinous membranes which may be floated out in water. Should 
calcification ultimately occur, hooklets may be found on rubbing up 
the material with water in a mortor. 

When suppuration takes place, polymorphonuclear leukocytes are 
first found between the cyst and its adventitious capsule; the cysts 
ultimately may become softened and burst, membranes, scolices, and 
hooklets floating about in the pus. (See also Sputum.) 



HYDRONEPHROSIS. 

The diagnosis of hydronephrosis can usually be made without diffi- 
culty if a sufficient amount of fluid can be obtained; the presence of 
urea and uric acid in notable quantities, as well as of renal epithelial 
cells, which latter especially should be sought for, is quite character- 
istic. Small amounts of uric acid, however, may also be present in 
ovarian cysts. 

PANCREATIC CYSTS. 

These cysts may be recognized by the fact that the fluid possesses 
the power of digesting albumin in alkaline solution. A small amount 
of the liquid is added to a few c.c. of milk, when after precipitation 
of the casein the biuret test is applied ; a positive reaction indicates 
the presence of trypsin. Unfortunately, however, the test does not 
always yield positive results, even if the fluid in question is derived 
from a pancreatic cyst, as the trypsin is apparently destroyed in the 
course of time. The larger the cyst, the less likely will it be pos- 
sible to obtain the reaction. A positive result is hence only of value, 
while a negative result does not exclude the existence of the disease. 1 

1 Karewski, Deutsch. med. Woch., 1890, vol. xvi, pp. 1035 and 1069. Hof- 
meister, Prag. med. Woch., 1891, vol. xvi, pp. 365 and 377 (see Gussenbauer). 
v. Jaksch, Zeit. f. Heilk., 1888, vol. ix, p. 126 (see Wolfler). 



CHAPTER XL 

THE SEMEN. 

The ejaculated semen is a mixture of the secretions furnished by 
the testicles, the prostate gland, the seminal vesicles, and the glands 
of Cowper. 

GENERAL CHARACTERISTICS. 

Semen is white or slightly yellowish in color, semifluid, sticky, 
and of an opaque, non-homogeneous, milky appearance, which is due 
to the presence of white, opaque islets floating in the otherwise clear 
fluid; these consist almost entirely of the specific morphological 
elements of the semen, the spermatozoa. Its odor, which strongly 
resembles that of fresh glue, is characteristic, and is owing to the 
presence of spermin. It is generally attributed to an admixture of 
prostatic fluid, as the semen obtained from the vasa deferentia is 
odorless. According to Robin, however, this odor is produced only 
at the moment of ejaculation, and cannot be ascribed to any single 
one of the secretions present. The reaction of human semen is 
slightly alkaline, and its specific gravity greater than that of water, 
in which it sinks to the bottom. 



CHEMISTRY OF THE SEMEN. 

Accurate analyses of human semen or of mammalian semen do 
not exist, and only the old analyses of Vauquelin and Kolliker can 
be given : 

Man. Horse. Ox. 

Water 90 81.90 82.10 

Albuminous material ) f .... 15.30 

Extractives . . . \ 6 \ 16.45 

Ethereal extract . ) I 2 . 20 

Mineral material 4 1.61 2 . 60 

The mineral matter consists largely of calcium phosphate. 

If semen is kept, or if it is slowly evaporated, crystals of phos- 
phate of spermin separate out, which are commonly known as Bott- 
cher's crystals, and which were long regarded as identical with the 
so-called Charcot-Leyden crystals that are found in the sputum of 
bronchial asthma, in the blood of leukemia, in the stools in cases of 
helminthiasis, etc. 



624 THE SEMEN 

Spermin is a basic substance, and, according to Ladenburg and 
Abel, is closely related to, if not identical with, diethylene diamin 
(piperazin) : 

/NH X 
C 2 H 4 \ )C 2 H 4 

The phosphate crystallizes in the form of monoclinic four-sided 
spindles or prisms, which appear as flattened needles of variable 
size. Some are scarcely visible even with a fairly high power of the 
microscope, while others attain the length of 40 fx to 60 fJ-. The sub- 
stance is soluble in formalin, thus differing from the Charcot-Leyden 
crystals. In water it dissolves with difficulty; it is slowly soluble in 
acids and alkalies, even in ammonia, while it is insoluble in alcohol, 
ether, chloroform, and dilute saline solution. Florence's reagent 
(see below) colors the crystals a bluish black. According to Cohn, 
the Bottcher crystals are formed exclusively in the prostate gland, 
the gland itself furnishing the basic component, while the necessary 
phosphoric acid is derived from other portions of the reproductive 
apparatus. 1 



MICROSCOPIC EXAMINATION OF THE SEMEN. 

Upon microscopic examination normal semen is seen to contain 
innumerable, actively moving, thread-like bodies, measuring from 
50 g to 60 [J. in length — the spermatozoa. These consist of an egg- 
shaped head, when seen from above, which is from 3 /J- to 5 {J- in 
length, the broader end being dircted anteriorly; a middle portion, 
4 [J. to 6 fJ- in length, with which the head is united by its smaller 
end; and a posterior piece or tail, into which the middle piece grad- 
ually fades (Fig. 170). 

In addition to the spermatozoa a few hyaline bodies are seen which 
are derived from the seminal vesicles; further, numerous small, pale 
granules of an albuminous nature (lecithalbumin), some testicular 
and urethral epithelial cells, lecithin corpuscles, and so-called pros- 
tatic or amyloid corpuscles, which at first sight resemble starch 
granules in appearance, owing to their concentric striations. A few 
leukocytes and occasionally a few red corpuscles may also be found. 



PATHOLOGY OF THE SEMEN. 

The study of the semen has received little attention from clin- 
icians, and gynecologists frequently hold the wife responsible for 

1 Th. Cohn, "Zur Kenntniss d. Spermas," Centralbl. f. allg. Path. u. path. Anat., 
vol. x, pp. 940 and 949. 



THE RECOGNITION OF SEMEN IN STAINS 625 

sterility when an examination of the husband's semen would — 
according to Kehrer, 1 in 40 per cent. — reveal an absence of sperma- 
tozoa, constituting the condition usually spoken of as azoospermatism. 
This may be temporarily observed following venereal excesses, when 
the fluid finally ejaculated is almost entirely of prostatic origin; 
their absence then possesses no significance, but persistent azoosper- 
matism must of necessity be associated with sterility. 2 

Cases have been recorded in which, notwithstanding the presence 
of spermatozoa and apparently normal sexual conditions in both 
husband and wife, sterility existed nevertheless, but in which it was 
observed that the spermatozoa lost their motile power almost imme- 
diately after ejaculation. Under normal conditions, following inter- 
course actively moving spermatozoa may be found in the vagina 
after hours, days, and even weeks. 

Whenever it is deemed advisable to make an examination of the 
semen, this should be done immediately following ejaculation, or as 
soon as possible thereafter. The material should be placed in a test 
tube and this immersed in lukewarm water until it can be examined. 
Note should then be taken, not only of the presence, but also of the 
degree of motility of the spermatozoa, a drop of the semen being 
examined directly with the microscope. 

Bloody semen, constituting the condition spoken of as hemo- 
spermia, has been observed on several occasions. It may follow 
excessive sexual indulgence, but may also occur in connection with 
gonorrheal epididymitis. The blood is readily recognized upon micro- 
scopic examination. 3 



THE RECOGNITION OF SEMEN IN STAINS. 

In medicolegal cases the physician may be called upon to decide 
whether or not certain stains on body-linen are caused by spermatic 
fluid, whether or not a rape has been committed, etc. In such cases 
it is frequently only necessary to examine a drop of the vaginal 
fluid in order to arrive at a positive result at once. At other times 
recourse must be had to the following method : A fragment of the 
linen or scrapings from the vulva or vagina are placed in a watch- 
crystal and allowed to soak for at least one hour in from 27 to 30 
per cent, alcohol, when a bit of the material is teased in a solution 
of eosin in glycerin (1 to 200), and examined. The heads of the 
spermatozoa are thus stained a deep red, while the tails, which are 
often broken, exhibit a pale-rose tint, and can readily be distinguished 
from vegetable fibers, which do not take the stain at all. A positive 

1 Beitrage z. klin. u. exper. Gynak., 1879, vol. ii, Giessen. 

2 Fiirbringer, Zeit. f. klin. Med., 1881, vol. iii, p. 310. 

3 Feleki, Centralbl. f. Krankh. d. Harn- u. Sexualorgane, 1901, vol. xii, p. 506. 

40 



626 THE SEMEN 

statement can thus be made in every case, even after months and 
years, as spermatozoa not only resist the action of reagents, but also 
the process of putrefaction; this is probably owing to the large pro- 
portion of mineral matter which enters into their composition, and 
which ensures the preservation of their form. Instances have been 
recorded in which it was possible to demonstrate spermatozoa in 
stains after eighteen years. 

The semen test of Florence 1 has attracted much attention, and 
may be recommended in doubtful cases; only a negative result, 
however, is of value (see below). It is based upon the observation 
that very characteristic crystals of iodospermin are formed when 
spermatic fluid is treated with a solution of iodopotassic iodide 
containing 1.65 grams of pure iodine and 2.54 grams of potassium 
iodide, dissolved in 26 c.c. of water. When a drop of this solution 
is added to a drop of spermatic fluid or an aqueous extract of a seminal 
stain, dark-brown crystals of iodospermin separate out at once, and 
may be readily recognized under the microscope. They occur in 
the form of long rhombic platelets or fine needles, often grouped in 
rosettes, but also occurring singly or as twin crystals. The exami- 
nation with the microscope should be made at once after the addition 
of the reagent, as the crystals disappear on standing. 

As the reaction may also be obtained in cases of azoosperma- 
tism, and with pure prostatic secretion, while a negative result is 
obtained with the fluid from spermatoceles, it is manifest that the 
test is not applicable for the determination of the presence or ab- 
sence of spermatozoa per se. Posner 2 states that he obtained similar 
crystals when the test was applied to a glycerin extract of ovaries. 

More recently Richter 3 has shown that Florence's reaction is also 
obtained with a decomposition product of lecithin, viz., cholin, which 
would explain the observation that better results are commonly 
obtained with dried semen than with fresh material. But it follows 
also that the reaction cannot be a specific semen reaction, and Richter 
accordingly concludes that a negative result only is of value, and 
indicates that the material under examination is not semen. He states 
that he obtained positive results with vaginal and uterine mucus, 
with decomposing brain substance, and other organs as well. In 
confirmation of Richter's results, Bocarius 4 has demonstrated that 
the so-called iodospermin is in reality an iodized product of cholin 
and not of spermin. 

1 Du sperme et des taches de sperme en medecine legale, Arch. d'Anthrop. 
crimin., vols, x and xi. 

2 "Die Florence' sche Reaktion," Berlin, klin. Woch., 1897, p. 602. 

3 "D. mikrochemische Nachweis v. Sperma," Wien. klin. Woch., 1897, p. 569. 

4 Zeit. f. phys. Chem., 1902, vol. xxxiv, p. 339. 



CHAPTER XII. 
VAGINAL DISCHARGES. 

GENERAL CHARACTERISTICS. 

The secretion which is normally furnished by the vaginal glands 
is small in amount, and just sufficient to keep the mucous mem- 
brane moist. It is a clear or somewhat milky-looking, semiliquid 
material, in which numerous epithelial laminse may be found. It 
has been stated that the reaction of the vaginal secretion in virgins is 
invariably acid, while an alkaline reaction is the rule in the deflorees. 
During pregnancy, however, the secretion is probably always acid. 
In 500 cases which Kronig examined in this direction an alkaline 
reaction was never observed. According to Zweifel, 1 the vaginal 
secretion contains traces of trimethylamin, to which its peculiar odor 
is probably due. 

Microscopically, numerous epithelial cells, mucous corpuscles, a 
few large mononuclear leukocytes, cellular detritus, and bacteria 
are found. Doderlein 2 has described a non-pathogenic bacillus 
or a group of bacilli which are characterized by the fact that they 
give rise to marked acid fermentation of sugar, and he regards these 
organisms as the only ones which are constantly present in the nor- 
mal vagina. Kronig and Menge, however, state that they are often 
absent. These observers have found, on the other hand, that under 
normal conditions there are various bacilli and cocci present which 
belong to the class of obligatory anaerobes, and are likewise non- 
pathogenic. Unfortunately they have not described these organisms 
in detail. Near the outlet they found bacteria which may be culti- 
vated upon alkaline aeorbic culture media, but which are usually 
absent in the upper portion of the vagina. 

It is important to note that various diplococci may also be found 
under normal conditions, and care should be taken not to confound 
these with gonococci. Like the gonococci, they are decolorized by 
Gram's method. If the characteristics of the former be borne in 
mind, however, mistakes may probably always be avoided; in mar- 
ried women and in children it is best to make the diagnosis of gon- 
orrhea only when the gonococcus has been isolated by cultivation. 

The question whether or not pathogenic bacteria may occur in 
the normal vagina of pregnant or non-pregnant women, may be 

1 Arch. f. Gynak., 1881, vol. xviii, p. 359. 

2 Ibid., 1887, vol. xxxi, p. 412. 



628 VAGINAL DISCHARGES 

answered in the affirmative; but with the exception of the gono- 
coccus they are not often seen. 1 Bergholm 2 thus examined the 
vaginal secretion of 40 pregnant women, and was unable to obtain 
organisms pathogenic for animals in a single case. There were no 
pyogenic staphylococci, no streptococci, and no colon bacilli. 

The vaginal secretion has been shown to possess powerful bac- 
tericidal properties, so that pathogenic organisms, even when arti- 
ficially introduced into the vagina, are rapidly killed. Kronig thus 
found that the Bacillus pyocyaneus disappears from the vagina of 
pregnant women in from ten to thirty hours, the s aphylococci in 
from six to thirty-six hours, and the Streptococcus pyogenes within 
six hours. Important from a practical standpoint is the fact that 
the bacteria disappeared less rapidly when irrigation of the vagina 
with water or even antiseptics was employed. 

Of animal parasites, the Trichomonas vaginalis is occasionally 
encountered in the vaginal discharge. The organism is identical 
with the trichomonas found in the feces and in the urine. In the 
United States it is not so common as among the peasant population 
of Central Europe. As far as is known, the organism is of no patho- 
logical significance. From a medicolegal standpoint, however, its 
presence may not be unimportant, as cases are on record in which 
trichomonades have been confounded with spermatozoa. In my 
judgment, however, such a mistake can only occur if the observer 
is totally without training in microscopy. 

The possible presence of the Anguillula aceti in the vaginal dis- 
charge has been pointed out by Billings, Miller, and Stiles. Stiles 
has suggested that it may be introduced into the vagina by injections 
of vinegar-water taken with the object of preventing conception. 

VAGINAL BLENNORRHEA. 

In physiological conditions an increased vaginal secretion is ob- 
served during sexual excitement, just preceding and at the beginning 
of menstruation, and during pregnancy, when a profuse blennorrhea 
is frequently seen, which sometimes assumes a virulent character. 
The secretion under such conditions readily becomes purulent. 
When not dependent upon a gonorrheal infection the secretion is 
thicker than normal, white, and creamy. At times also the vaginal 
catarrh observed in pregnancy is complicated with mycosis, when 
white or yellowish-gray patches may be seen at the orifice of the 
vagina; the latter may, indeed, be filled with particles which consist 
entirely of fungi. 

1 Doderlein, Das Scheidensecret, Leipzig, 1892. J. W. Williams, Amer. Jour. 
Obstet., 1898, vol. xxxviii; Trans. Amer. Gyn. Soc, 1898; Amer. Jour. Obstet., 
1898. 

2 Arch. f. Gynak., 1902, vol. lxvi, Heft 3. 



VULVITIS AND VAGINITIS 629 



MENSTRUATION. 

At the beginning of menstruation, as has been pointed out above, 
an increase in the amount of vaginal secretion is observed, in which 
leukocytes, prismatic epithelial cells coming from the uterus, as well 
as the usual vaginal cells, may be seen upon microscopic exami- 
nation. Later the secretion becomes sanguineous in character, and 
finally only epithelial cells, leukocytes, and granular detritus are 
encountered, the cells usually showing evidence of fatty degenera- 
tion. The amount of blood lost at each menstrual period amounts 
to about 200 grams in perfectly healthy females. 

THE LOCHIA. 

The lochia during the first day following parturition are red in 
color — the lochia rubra — and emit the characteristic sanguineous 
odor. At this time a microscopic examination will reveal an abun- 
dance of red corpuscles,, some leukocytes, and a variable number 
of epithelial cells, which are almost exclusively of vaginal origin. 
On the second and third days the number of red corpuscles dimin- 
ishes, while the leukocytes increase in number. Still later the dimi- 
nution in the red and the increase in the white corpuscles become 
more marked, and the discharge at the same time assumes a grayish 
or white color, until about the tenth day the red corpuscles have 
almost entirely disappeared, while the leukocytes and epithelial cells 
are abundant. Finally, the secretion becomes thicker, mucoid, and 
milky white in color — the lochia alba — which condition may persist 
for from three to four weeks in nursing women, and still longer in 
those who do not nurse, until finally the normal secretion is again 
established. Numerous bacteria are encountered in the lochia, and 
it is curious to note that among these pus organisms are quite con- 
stantly present without giving rise to symptoms. When a portion 
of the placenta or membranes have been retained the lochia soon 
give off a fetid odor, and assume a dirty brownish color; the reten- 
tion of blood clots alone may also produce this result. In such cases 
the lochia swarm with bacteria of all kinds. 1 

VULVITIS AND VAGINITIS. 

In cases of vulvitis and vaginitis a marked increase is observed 
in the number of the leukocytes and epithelial cells, the character of 
the latter depending essentially, of course, upon the portion of the 
genital tract affected. Red corpuscles are also met with at times; 
their number generally stands in a direct relation to the intensity of 

1 Doderlein, loc. cit. Thomen, Centralbl. f. d. med. Wiss., 1890, vol. xxviii, 
p. 537; and Arch. f. Gyn., 1889, vol. xxxvi, p. 231. 



630 



VAGINAL DISCHARGES 



the inflammatory process. In some instances epithelial casts of 
the entire vagina have been observed, constituting the condition 
termed vaginitis exfoliativa. The condition, however, is rare. 
In mycotic vaginitis leptothrices have been found by v. Herff. 1 
The discharge of large amounts of pure pus through the vagina 
points to perforation of an abscess of the genital organs or of the 
neighboring structures into the uterus or the vagina; it is of rare 
occurrence. Much more common is the discharge of fecal matter, 
or of urine through this channel, indicating the existence of a vagino- 
rectal or vaginovesical fistula. 



MEMBRANOUS DYSMENORRHEA. 

While ordinarily, during menstruation, shreds of desquamated 
uterine lining are frequently encountered, it is rare to meet with 
large pieces or complete casts of the uterus, the elimination of which 
is usually associated with the symptoms of a severe dysmenorrhea, 
constituting the condition spoken of as membranous dysmenorrhea. 

CANCER. 

While the diagnosis of malignant growth of the uterus is probably 
never based upon a microscopic examination of the vaginal discharge 
alone, it may be mentioned that in advanced cases this is possible, 
as fragments of an epithelioma of the cervix, for example, may 
frequently be detected upon microscopic examination. In sus- 
pected cases small pieces of tissue should be excised and examined 
according to usual histological methods. 2 



GONORRHEA. 

In suspected cases of gonorrhea an examination of the vaginal 
and urethral discharge for the presence of gonococci is important, 
as it is practically impossible to diagnosticate this condition positively 
in any other manner. Care should be taken, however, not to con- 
found the diplococci which may be normally present in the urethra 
and vagina with gonococci. (See chapter on the Urine.) Unfortunately, 
however, excepting in fairly acute cases, these examinations are 
rather unsatisfactory. There can be no doubt that in many cases, 
which unquestionably are gonorrheal, the ordinary microscopic 
examination is negative. Better results may possibly be reached if 
the examination is made twenty-four hours following the injection of 
gonococcus vaccine (dose, 10,000,000 organisms). 

1 Centralbl. f. Bakter., 1895, p. 750. 

2 T. S. Cullen, Cancer of the Uterus, Appleton & Co., 1900. 



ABORTION 



631 



ABORTION. 

In cases of abortion it is often possible to discover chorion villi in 
the expelled blood-clots which present the characteristic capillary 
network (Fig. 174), and often manifest signs of advanced fatty 




Fig. 174.— Chorion villi. 




Fig. 175. — Decidual cells. 



degeneration. Important also from a diagnostic point of view is the 
presence of decidual cells (Fig. 175), which are characterized by their 
large size, their round, polygonal, or spindle-like form, and their 
characteristic nuclei and nucleoli. 



CHAPTER XIII. 

THE SECRETION OF THE MAMMARY GLANDS. 

THE SECRETION OF MILK IN THE NEWBORN. 

A secretion from the mammary glands of the male is observed 
only in the newborn, if we except those rare cases in which adult 
males were known to suckle infants. The fluid in question, which 
may also be obtained from the female infant, is termed "Hexenmilch" 
(witches' milk) by the Germans. Qualitatively it has the same com- 
position as milk, but may manifest considerable quantitative varia- 
tions. 

COLOSTRUM. 

Aside from those curious instances in which a secretion of milk 
has been observed in non-pregnant women, mammary activity is 
essentially connected with the physiological phenomena of pregnancy 







?©?•' 

Fig. 176. — Colostrum of a woman in sixth month of pregnancy. (Eye-piece III, 
obj. 8 A, Reichert.) (v. Jaksch.) 

and parturition. Often as early as the third month a small drop of 
a serous-looking fluid can be obtained from the nipple by pressure 
upon the breasts. Immediately after delivery a variable amount of 
fluid is secreted, which is watery, semi-opaque, mucilaginous, and 
of a yellowish color. To this secretion, as well as to that observed 
during pregnancy, the term colostrum has been applied. It is dis- 
tinguished from true milk by its physical characteristics and by the 
presence of a greater proportion of sugar and salts. The fluid, 
moreover, coagulates upon boiling. An idea may be formed of its 
composition from the appended table: 



HUMAN MILK 



633 









4 weeks before birth. 


17 days be- 
fore birth. 


9 days be- 
fore birth. 


24 hours 
after birth. 


2 days 




I. 


II. 


after birth. 


Water 


945.2 


852.0 


851.7 


858.8 * 


843.0 


867.9 


Solids . 






54.8 


148.0 


148.3 


141.2 


157.0 


132.1 


Casein 


















21.8 


Albumin 






28.8 


69.0 


74.8 


80.7 






Fat . 






7.3 


41.3 


30.2 


23.5 




48.6 


Lactose 






17.3 


39.5 


43.7 


36.4 




61.0 


Salts . 






4.4 


4.4 


4.5 


5.4 


5.1 





Upon microscopic examination fat droplets, a few leukocytes, 
some epithelial cells, and so-called colostrum corpuscles are found. 
The latter are highly refractive bodies, of irregular size, whose inte- 
rior is filled with fatty granules (Fig. 176). 

Literature. — G. Woodward, Jour. Exper. Med., vol. ii, p. 217. 

THE SECRETION OF MILK PROPER, IN THE ADULT FEMALE. 

The secretion of milk proper usually begins about the third day 
following parturition, and may continue for a variable length of 
time. On the one hand, the amount of milk secreted may be so 
small as to be insufficient for the needs of the child, so that lacta- 
tion may have to cease after several days; on the other hand, women 
are not infrequently seen who nurse their children for two years and 
even longer and who may furnish four liters a day. Usually infants 
are nursed until six or seven teeth have appeared, which period varies 
with the individual child, averaging about the eleventh month. 



HUMAN MILK. 

Human milk is of a bluish color, and differs in this respect from 
the milk of cows. Its reaction is alkaline. The specific gravity 
may vary between 1.026 and 1.035, one between 1.028 and 1.034 
being the most common. The amount of milk secreted in twenty- 
four hours varies from 500 to 1500 c.c. Microscopically, it is a 
fairly homogeneous emulsion of fat, and is practically destitute of 
cellular elements. From the following table an idea may be formed 
of its chemical composition : 





Biehl. 


Gerber. 


Christenn. 


Pfeiffer. 


Pfeiffer. 


Mendes de 
Leon. 


Water 


876.00 


891.00 


872.40 


892.00 


890.60 


877 . 90 


Solids .... 


124.00 


109.00 


127.60 


108.00 


109.40 




Albumin .... 


22.10 


17.90 


19.00 


16.13 


17.24 


25 . 30 1 


Fat ... . 


38 . 10 


33.00 


43.20 


32.28 


29.15 


38 . 90 ' 


Lactose . 


60.90 


53.90 


59.80 


57.94 


59.92 


55 . 40 


Salts 


2.90 


4.20 


2.60 


1.65 


2.09 


2.50j 



634 THE SECRETION OF THE MAMMARY GLANDS 

Upon comparing this table with the following analysis of cows' 
milk it will be seen that the latter contains more albumin and less sugar 
than human milk. Human milk, moreover, is relatively deficient in 
mineral matter, and especially in calcium salts and phosphoric acid: 

Water 874.2 

Solids 125.8 

Casein . . • 28.8 \ Q/) , 

Albumin 5.3/ d4i) 

Fat 36.6 

Lactose 48 . 1 

Salts 7.1 

Of inorganic salts human milk contains about 0.7 pro mille of 
potassium (K 2 0), 0.2 of sodium, 0.3 of calcium, 0.06 of magnesium, 
from 3.52 to 7.21 mgrms. of iron, about 0.4 pro mille of phosphoric 
acid, and 0.4 of chlorine., 

The albumins found in milk plasma are casein, lactoglobulin, 
and lactalbumin. It is claimed by some observers that the casein 
of human milk differs from that obtained from cows' milk. The 
casein coagula in human milk are not so large and dense as those 
observed in cows' milk. Human casein, moreover, is not so readily 
precipitated by acids and salts; it does not always coagulate upon 
the addition of rennet ferment, and while it may be precipitated by 
the gastric juice, it is readily dissolved by an excess. 

The question whether or not normal human milk contains micro- 
organisms may now be answered in the affirmative. There can be 
no doubt, however, that the milk as it is secreted by the healthy 
gland is sterile, but upon passing along the lacteal ducts in the 
nipple it is always contaminated by the Staphylococcus epidermidis 
albus (Welch). This microorganism must be regarded as a constant 
inhabitant of the skin, and is the only one of the cutaneous bacteria 
which penetrates the deeper layers of the epidermis and the gland- 
ular appendages of the skin. It is thus apparent why this organism 
is so constantly met with, and is practically the only one found in 
normal human milk. Exceptionally the Staphylococcus pyogenes 
aureus is found. 

THE MILK IN DISEASE. 

The chemistry of the milk in pathological conditions has received 
little attention. It appears, however, that the milk of women when 
ill usually contains less fat, and that the proportion of lactose is 
diminished. In cases of jaundice the presence of bile pigment and 
of biliary acids has not been satisfactorily demonstrated. According 
to Friedjung, 1 a subnormal amount of iron is usually found in the 
milk when nurslings do not thrive on apparently normal milk. In 

1 Arch. f. Kinderheilk., vol. xxxi, Heft 1 u, 2. 



THE MILK IN DISEASE 



635 



cases of mammary tumors bloody secretion has been 
observed in rare cases, the nipple itself being intact. 

Microscopically, an admixture of leukocytes is 
observed in various diseases of the breasts, and 
especially in cases of abscess. Of pathogenic 
microorganisms, streptococci may be found in cases 
of puerperal fever; more commonly, however, they 
are absent. The typhoid bacillus has occasionally 
been seen in cases of typhoid fever, and it is inter- 
esting to note that the specific agglutinins of typhoid 
fever have been found in the milk. Pneumococci 
have been obtained from the milk of pregnant 
women affected with lobar pneumonia. The im- 
portant question whether or not tubercle bacilli are 
eliminated in the milk in cases of phthisis cannot 
be definitely answered. In cows such an occur- 
rence is certainly common, even when there is no 
demonstrable tuberculous lesion of the udder. So 
far as I have been able to ascertain, however, 
tubercle bacilli have never been found in human 
milk. 1 

A blue and a red color have been observed in 
the milk of cows, owing to the presence of the 
Bacillus pyocyaneus and the Micrococcus pro- 
digiosus, respectively. 

A chemical examination of human milk should 
always be made when it is apparent that the nutri- 
tion of the baby is below normal. Valuable dietetic 
suggestions may thus be obtained. In other cases, 
as when the mother is unwilling or unable to nurse 
her child beyond a certain period, a knowledge 
of the composition of her milk: will enable the 
physician to give specific instructions regarding the 
proper modification of cows' milk. If a wet-nurse 
is to be employed, her milk should likewise be 
examined. Most important is the determination 
of the specific gravity and of the amount of fat. 
The former may vary between 1 .029 and 1 .033. The 
amount of fat should not be less than 3 per cent. 

Determination of the Specific Gravity.— The specific gravity is 
best determined with the lactodensimeter of Quevenne (Fig. 177). As 
the instrument is graduated for a temperature of 60° F., it is necessary 
to correct the specific gravity when the temperature is above or below 

1 Escherich, Fortschr. d. Med., 1885, vol. iii, p. 321. Karlinski, Wien. med. 
Woch., 1888, vol. xxxviii, No. 28. Ott, Prag. med. Woch., 1892, vol. xvii, p. 
145. Cohn u. Neumann, Virchow's Archiv, 1880, vol. cxxvi, p. 187. 




Fig. 177 



Quevenne's 
lactodensimeter. 



636 



THE SECRETION OF THE MAMMARY GLANDS 



this point. In the following tables the corrected specific gravity may 
be found corresponding to temperatures ranging from 46° to 75° F. : 

Corrections for Temperature. 









Degrees of thermometer (Fahrenheit). 






Specific gravity. 






















46 


47 


48 


49 


50 


51 


52 


53 


54 


55 


1020 


19.0 


19.1 


19.1 


19.2 


19.2 


19.3 


19.4 


19.4 


19.5 


19.6 


1021 


20.0 


20.0 


20.1 


20.2 


20.2 


20.3 


20.3 


20.4 


20.5 


20.6 


1022 


21.0 


21.0 


21.1 


21.2 


21.2 


21.3 


21.3 


21.4 


21.5- 


21.6 


1023 


22.0 


22.0 


22.1 


22.2 


22.2 


22.3 


22.3 


22.4 


22.5 


22.6 


1024 


22.9 


23.0 


23.1 


23.2 


23.2 


23.3 


23.3 


23.4 


23.5 


23.6 


1025 


23.9 


24.0 


24.0 


24.1 


24.1 


24.2 


24.3 


24.4 


24.5 


24.6 


1026 


24.9 


24.9 


25.0 


25.1 


25.1 


25.2 


25.2 


25.3 


25.4 


25.5 


1027 


25.9 


25.9 


26.0 


26.1 


26.1 


26.2 


26.2 


26.3 


26.4 


26.5 


1028 


26.8 


26.8 


26.9 


27.0 


27.0 


27.1 


27.2 


27.3 


27.4 


27.5 


1029 


27.8 


27.8 


27.9 


28.0 


28.0 


28.1 


28.2 


28.3 


28.4 


28.5 


1030 


28.7 


28.7 


28.8 


28.9 


29.0 


29.1 


29.1 


29.2 


29.4 


29.4 


1031 


29.6 


29.6 


29.7 


29.8 


29.9 


30.0 


30.1 


30.2 


30.3 


30.4 


1032 


30.5 


30.5 


30.6 


30.7 


30.9 


31.0 


31.1 


31.2 


31.3 


31.4 


1033 


31.4 


31.4 


31.5 


31.6 


31.8 


31.9 


32.0 


32.1 


32.3 


32.4 


1034 


32.3 


32.3 


32.4 


32.5 


32.7 


32.9 


33.0 


33.1 


33.2 


33.3 


1035 


33.1 


33.2 


33.4 


33.5 


33.6 


33.8 


33.9 


34.0 


34.2 


34.3 









Deg 


rees of thermometer (Fahrenh 


eit). 






Specific gravity. 






















56 


57 


58 


59 


60 


61 


62 


63 


64 


65 


1020 


19.7 


19.8 


19.9 


19.9 


20.0 


20.1 


20.2 


20.2 


20.3 


20.4 


1021 


20.7 


20.8 


20.9 


20.9 


21.0 


21.1 


21.2 


21.3 


21.4 


21.5 


1022 


21.7 


21.8 


21.9 


21.9 


22.0 


22.1 


22.2 


22.3 


22.4 


22.5 


1023 


22.7 


22.8 


22.8 


22.9 


23.0 


23.1 


23.2 


23.3 


23.4 


23.5 


1024 


23.6 


23.7 


23.8 


23.9 


24.0 


24.1 


24.2 


24.3 


24.4 


24.5 


1025 


24.6 


24.7 


24.8 


24.9 


25.0 


25.1 


25.2 


25.3 


25.4 


25.5 


1026 


25.6 


25.7 


25.8 


25.9 


26.0 


26.1 


26.2 


26.3 


26.5 


26.6 


1027 


26.6 


26.7 


26.8 


26.9 


27.0 


27.1 


27.3 


27.4 


27.5 


27.6 


1028 


27.6 


27.7 


27.8 


27.9 


28.0 


28.1 


28.3 


28.4 


28.5 


28.6 


1029 


28.6 


28.7 


28.8 


28.9 


29.0 


29.1 


29.3 


29.4 


29.5 


29.6 


1030 


29.6 


29.7 


29.8 


29.9 


30.0 


30.1 


30.3 


30.4 


30.5 


30.7 


1031 


30.5 


30.6 


30.8 


30.9 


31.0 


31.2 


31.3 


31.4 


31.5 


31.7 


1032 


31.5 


31.6 


31.7 


31.9 


32.0 


32.2 


32.3 


32.5 


32.6 


32.7 


1033 


32.5 


32.6 


32.7 


32.9 


33.0 


33.2 


33.3 


33.5 


33.6 


33.8 


1034 


33.5 


33.6 


33.7 


33.9 


34.0 


34.2 


34.3 


34.5 


34.6 


34.8 


1035 


34.5 


34.6 


34.7 


34.9 f 


35.0 


35.2 


35.3 


35.5 


35.6 


35.8 



Specific gravity. 


Degrees of thermometer (Fahrenheit). 
























66 


67 


68 


69 


70 


71 


72 


73 


74 


75 


1020 


20.5 


20.6 


20.7 


20.0 


21.0 


21.1 


21.2 


21.3 


21.5 


21.6 


1021 


21.6 


21.7 


21.8 


22.0 


22.1 


22.2 


22.3 


22.4 


22.5 


22.6 


1022 


22.6 


22.7 


22.8 


23.0 


23.1 


23.2 


23.3 


23.4 


23.5 


23.7 


1023 


23.6 


23.7 


23.8 


24.0 


24.1 


24.2 


24.3 


24.4 


24.6 


24.7 


1024 


24.6 


24.7 


24.9 


25.0 


25.1 


25.2 


25.3 


25.5 


25.6 


25.7 


1025 


25.6 


25.7 


25.9 


26.0 


26.1 


26.2 


26.4 


26.5 


26.6 


26.8 


1026 


26.7 


26.8 


27.0 


27.1 


27.2 


27.3 


27.4 


27.5 


27.7 


27.8 


1027 


27.7 


27.8 


28.0 


28.1 


28.2 


28.3 


28.4 


28.6 


28.7 


28.9 


1028 


28.7 


28.8 


29.0 


29.1 


29.2 


29.4 


29.5 


29.7 


29.8 


29.9 


1029 


29.8 


29.9 


30.1 


30.2 


30.3 


30.4 


30.5 


30.7 


30.9 


31.0 


1030 


30.8 


30.9 


31.1 


31.2 


31.3 


31.5 


31.6 


31.8 


31.9 


32.1 


1031 


31.8 


32.0 


32.2 


32.2 


32.4 


32.5 


32.6 


32.8 


33.0 


33.1 


1032 


32.9 


33.0 


33.2 


33.3 


33.4 


33.6 


33.7 


33.9 


34.0 


34.2 


1033 


33.9 


34.0 


34.2 


34.3 


34.5 


34.6 


34.7 


34.9 


35.1 


35.3 


1034 


34.9 


35.0 


35.2 


35.3 


35.5 


35.6 


35.8 


36.0 


36.1 


36.3 


1035 


35.9 


36.1 


36.2 


36.4 


36.5 


36.7 


36.8 


37.0 


37.2 


37.3 



PLATE XXIII. 



f-7 

|» 3 
■?-2 




Babeoek Flask, showing Fat in Neck. (Harrington.) 



THE MILK IN DISEASE 637 

Estimation of the Fat. — The estimation of the fat is most con- 
veniently accomplished with the aid of the Babcock apparatus. Special 
tubes (Plate XXIII) which have a long, graduated neck accompany 
the instrument. With a special pipette 17.6 c.c. of milk are measured 
off and introduced into one of the flasks. To this is added an equal 
volume of concentrated sulphuric acid (specific gravity 1.8), which 
should be slowly done, agitating the mixture gently by a rotary 
motion. The flask is placed in the centrifugal machine (counter- 
balanced by a flask, similarly weighted) and whirled for five minutes. 
After this boiling water is added to the base of the neck of the flask 
and the mixture centrifugated for three more minutes; boiling water 
is then further added until the layer of fat is well within the neck of 
the bottle and centrifugation continued for two to three minutes 
longer. The percentage of fat is finally read off directly on the neck 
of the bottle. If it should happen that the fat is caked in the tube, 
this is placed in hot water and melted, after which the reading is 
taken. 

For human milk Leffman and Bean have modified these bottles. 
They are graduated so that each small division corresponds to 0.3 
per cent, of fat. The sulphuric acid is of the same strength as above, 
but the amount of milk which is needed is only 2.92 c.c, which is 
measured off by means of a special pipette. This is treated with an 
equivalent volume of sulphuric acid, after the previous addition of 
0.6 c.c. of a mixture of equal parts of concentrated HC1 and amyl 
alcohol. Special jackets are provided into which the test bottles 
fit, and these can be attached to the common laboratory centrifuge 
used in urine work. 

Estimation of Lactose, — The lactose may be estimated polarimetri- 
cally or as follows: Dilute 10 c.c. of milk to 50 c.c. with water and 
add dilute acetic acid carefully until all the casein has separated out. 
Filter and wash the precipitate with water until the total bulk of the 
filtrate is 100 c.c. Boil in order to remove the coagulable albumins; 
filter again, wash through the filter until the filtrate is again brought 
to 100 c.c. In this final solution determine the amount of lactose 
by Fehling's method (which see). 10 c.c. of Fehling's solution 
require 0.067 gram of lactose for the complete reduction of the copper. 

Estimation of the Proteids. Boggs' Method. 1 — This is the most 
satisfactory for routine work and can be warmly recommended. It 
is based upon the precipitation of the proteids with phosphotungstic 
acid in hydrochloric acid solution, and in Esbach tubes. 

The reagent is prepared as follows: 25 grams of phosphotungstic 
acid are dissolved in 125 c.c. of distilled water. To this are added 
concentrated hydrochloric acid 25 c.c, diluted with distilled water 
100 c.c. This is essentially a 10 per cent, solution of phosphotungstic 

1 Johns Hopkins Hosp. Bull., vol. xvii, October, 1906. 



638 THE SECRETION OF THE MAMMARY GLANDS 

acid in 3 per cent. HC1; it is said to keep for months, when kept in 
a dark bottle. 

The Esbach tubes which are used are the common ones, reading 
from 1 to 7 grams pro liter; those reading to 12 gave unsatisfactory 
results. 

The milk is diluted to 1 in 10 for human milk and 1 in 20 for cows' 
milk; if the proteid content is very low a dilution to 1 in 5, viz., 1 in 
10 will answer. 

The diluted milk is poured into the tube to the mark U and the 
reagent added to R, reading the bottom of the meniscus. After clos- 
ing with a stopper the tube is inverted a dozen times and set aside in 
a vertical position for twenty-four hours. With dilutions of 1 in 10 the 
percentage is read off directly from the scale, while with a dilution 
of 1 in 20 we multiply by 2 and with one of 1 in 5 we divide by 2. 

It is essential that the dilutions should be accurate. A convenient 
outfit consits of a 2 c.c. pipette graduated in tenths and a small 
standard flask of 20 c.c. 



CHAPTER XIV. 

THE OPSONINS. 

The term opsonin has been introduced by Wright and Douglas to 
designate certain substances present in blood serum which render 
various bacteria subject to phagocytosis. The organisms in question 
are the various staphylococci, streptococci, pneumococci, meningo- 
cocci, gonococci, influenza bacilli, diphtheria and pseudodiphtheria 
bacilli, anthrax bacilli, tubercle, typhoid and colon bacilli, the plague 
bacillus, and others. For all these normal human blood serum con- 
tains opsonic material, but it is to be noted that with certain bacteria 
phagocytosis only occurs if the strains are not highly virulent. This 
is notably the case with streptococci and pneumococci. On the other 
hand, it should be borne in mind that with some organisms phago- 
cytosis will take place in normal salt solution, in the absence of 
blood serum (Bacillus pyocyaneus, Bacillus subtilis, and others). 

While normal opsonins are more or less thermolabile, being usually 
destroyed by heating for ten minutes at 60° C, the opsonins of immune 
sera are generally speaking more stable. Whether or not the immune 
opsonins are identical with the normal opsonins, as Wright claims, 
has not been definitely established. My own researches, in contra- 
distinction to those of Wright and Bulloch tend to show that the nor- 
mal opsonins are non-specific, while in infections results are at times 
obtained which suggest a certain specificity of the opsonic material. 

Of the chemical nature of the opsonins nothing is known. In 
association with Lamar, I have shown that they are intimately asso- 
ciated with the euglobulin fraction of the blood albumins, but there 
is some reason to think that they are carried down only mechanically 
with this fraction and that they are not necessarily globulins them- 
selves. 

Of the structure of the opsonins also nothing is known. Hektoen 
suggests that they may contain a haptophoric group which unites with 
bacterial or other receptors, and a functional opsoniferous group which 
brings about such changes in the cell as may be necessary to subject 
it to phagocytosis. 

Savtchenko and Dean view immune opsonins as amboceptors, but 
have not furnished a satisfactory basis for such an assumption. Greig- 
Smith regards the immune opsonins and agglutinins as identical, and 
looks upon the process of opsonification as the first phase of agglu- 
tination, but also, I think, without an adequate basis. 



640 THE OPSONINS 

Opsonins occur in all classes of vertebrates, and it is noteworthy 
that the serum of different animals (fish, frog, turtle, chicken, guinea- 
pig, rabbit, calf, sheep, pig, dog, cat, etc.) is capable of activating 
various microorganisms for phagocytosis by leukocytes of animals 
of different species. 

Besides bacteria other cells can also be opsonified. Barrath has 
noted the presence of opsonins in small amount for red cells in nor- 
mal serum (hemopsonins). Hektoen obtained similar results and 
also showed that blastomycetes from human lesions become sur- 
rounded by masses of leukocytes in the presence of normal human and 
dog serum. Preliminary observation (according to Hektoen) further 
indicates that phagocytosis of trypanosomes also is dependent upon 
opsonification. Savtchenko and Melkich obtained pronounced phago- 
cytosis of the spirochete of relapsing fever with serum of convales- 
cent patients. 

Clinical interest centres in Wright's observation that in certain 
bacterial infections the opsonins are frequently diminished, and that it 
is possible by means of bacterial vaccines to raise the opsonic value 
and thereby to increase the patient's resistance to the invading micro- 
organisms. As a matter of fact it has now been satisfactorily estab- 
lished that the bacterial vaccines represent a most important addition 
to our therapeutic armamentarium and that it is possible in many 
cases to cause either a cure or an improvement in the patient's condi- 
tion by means of such vaccines, where with older methods of treatment 
no result at all or only very slow improvement could be expected. 
This is notably the case in the more chronic bacterial infections, such 
as acne, sycosis, furunculosis, various types of tuberculosis (notably 
the so-called surgical forms), endocarditis, bacterial arthritis, unre- 
solved pneumonia, empyema, the most diverse wound infections, etc. 

The basis in the treatment of these various conditions, according 
to Wright, is the opsonic index, viz., the opsonic value of the infected 
individual as compared with the normal. According to his teachings 
the injection of a dose of vaccine is followed by a decrease of the 
opsonins (the negative phase), which is of variable degree and dura- 
tion depending upon the amount injected. This is followed by an 
increase (the positive phase), coincidently with which there is a 
corresponding improvement in the patient's condition. The idea is 
to so gauge and interspace the different doses that a negative phase 
is obviated as far as possible and a "high tide" of increased opsonic 
content secured. 

While a low opsonic value is the rule in the more chronic cases, and 
especially in connection with the more localized infections, high 
indices may be observed in acute cases with active systemic manifes- 
tations, and frequently alternate with low values. 

Deviations from the normal may at times be of distinct diagnostic 



THE OPSONINS 641 

value when they affect a particular microorganism, and Wright 
cites a number of examples to emphasize this point. I cite some 
of his more important deductions in the diagnosis of tuberculous 
infection : 

1. Conclusions which can be arrived at when we have at disposal 
the results of a series of measurements (opsonic determinations) : 

(a) When a series of measurements of the opsonic power of the blood 
reveals a persistingly low opsonic power with respect to the tubercle 
bacillus, it may be inferred, in the cases where there is evidence of a 
localized bacterial infection which suggests tuberculosis, that the 
infection in question is tuberculous in character. 

(b) When repeated examination reveals a persistingly normal 
opsonic power with respect to the tubercle bacillus, the diagnosis of 
tubercle may with probability be excluded. 

(c) When there is revealed by a series of blood examinations a con- 
stantly fluctuating opsonic index the presence of active tuberculosis 
may be inferred. 

2. Conclusions which may be arrived at where we have at disposal 
the result of an isolated blood examination: 

(a) When an isolated blood examination reveals that the tuber- 
culo-opsonic power of the blood is low, we may — according as we have 
evidence of a localized bacterial infection or of constitutional disturb- 
ance — infer with probability that we are dealing with tuberculosis — 
in the former case with a localized tuberculous infection, and in the 
latter with an active systemic infection. 

(b) When an isolated blood examination reveals that the tuberculo- 
opsonic power of the blood is high, we may infer that we have to deal 
with a systemic tuberculous infection which is active, or has recently 
been active. 

(c) When the tuberculo-opsonic power is found normal or nearly 
normal, while there are symptoms which suggest tuberculosis, we are 
not warranted, apart from the further test described below, in arriving 
at a positive or a negative diagnosis. 

The further criterion to which reference has been made in the pre- 
ceding paragraph is the following: 

When a serum is found to retain in any considerable measure, after 
it has been heated to 60° C. for ten minutes, its power of inciting pha- 
gocytosis we may conclude that "incitor elements" (immune opsonins) 
have been elaborated in the organism either in response to auto- 
inoculations occurring spontaneously in the course of tuberculous 
infection, or, as the case may be, under the artificial stimulus supplied 
by the inoculation of tubercle vaccine. 

The above considerations apply also in the case of other bacterial 
infections, and in the examination of exudates as well. 

In a general way my own observations bear out the correctness of 
Wright's diagnostic inferences, but I am inclined to attach importance 
41 



642 THE OPSONINS 

only to the results of repeated examinations and to pronounced devia- 
tions from the normal variations, viz., 0.8 to 1.2. Single observations 
are of relatively little importance, and purely localized infections 
without systemic symptoms may show no deviation from the normal 
whatever. Positive results are thus only of value, while normal 
values do not exclude the existence of infection. 

As regards the necessity of controlling bacterial inoculation by 
the opsonic index, my own observations and those of some of my col- 
leagues tend to show that the acts on this subject are by no means 
closed. If attempts at progressive active immunization are. to be 
made, an index of some kind is certainly desirable, aside from purely 
clinical symptoms, and the opsonic index in these cases is in my 
opinion unquestionably of value. If we see, as the result of repeated 
injections, that the phagocytic power of the patient is rapidly being 
diminished and finally practically held in abeyance, there can be no 
doubt to my mind that immunization in such a case is not being 
carried out to best advantage. The index in such cases would cer- 
tainly be of value. Then, again, if we find that a single inoculation in 
a given case invariably causes a marked drop in the phagocytic power, 
while smaller doses do not bring about this result, it is clear that here 
also the determination of the index would be of value. I should hence 
advocate its use in therapeutic work, especially in cases showing active 
disease, and notably so in children in whom a marked depression of the 
phagocytic power may be caused by using vaccine in the doses recom- 
mended for adults. 

In markedly chronic cases, on the other hand, in which the opsonic 
index shows but little variation from the normal, bacterial vaccines 
may safely be used in small doses and at intervals of from eight to 
ten days with but little control by the index. 

An opsonic immunity, in the sense of a continued high index as 
the result of immunization, does not exist. In the majority of cases 
the injection of a suitable dose of vaccine causes no negative phase 
which would not readily be explained by unavoidable errors of tech- 
nique ; after several days there is then a rise of a few tenths and after 
that a return to near the normal line. Continued high values are 
very rarely seen; sooner or later there is a return to normal, even 
though improvement continues. 



TECHNIQUE. 

Principle. — Wright's method is based upon the comparison of the 
number of organisms taken up by a given number of leukocytes under 
the opsonifying effect of the patient's serum, with the corresponding 
number observed in the case of a normal control serum, the latter 
value being placed as 1. 



TECHNIQUE 643 

Example. — Supposing that with the patient's serum the average 
number of organisms pro cell (the phagocytic index) was 5 and with 
the normal serum 10, then from the equation 10 : 1 : : 5 : x, it would 
follow that the opsonic index was 0.5. 

I have pointed out at another place that Wright's method is open 
to certain fallacies, and that more accurate results can be obtained by 
estimating the percentage of phagocyting leukocytes. By comparing the 
figures thus obtained with the figure corresponding to a specimen of 
normal blood serum, terming the latter value 1, an index is obtained 
which is directly comparable to Wright's index. As the percentage of 
phagocyting cells is to a certain extent dependent on the number of 
organisms present, it is advantageous to work with an emulsion 
which with normal serum will give a percentage of about 50; this 
will allow for an increase of the index in the patient's blood to 
about 2, which is sufficient for all practical purposes. If higher 
values are to be expressed it is necessary to dilute the serum (both 
normal and that of the patient) in the proportion of 1 to 10, 1 to 
20, etc., with saline solution and to proceed upon the same prin- 
ciple. 

Example. — With the patient's serum 80 per cent, of the cells were 
found to be phagocyting and with normal serum only 50. The index 
would be obtained according to the equation 50 : 1 : : 80 : x, and would 
accordingly be 1.6. 

If for any reason Wright's bacillary index is to be used, I should 
recommend that the percentage index be calculated at the same time; 
it will be found a useful check upon the former and readily shows up 
errors that may have been made in counting, depending on clumping 
of the organism. Under favorable conditions both will agree to the 
second decimal. If both are normal, above or below normal, Wright's 
index may be regarded as giving more or less correct values, but if 
they differ, the one being high and the other low, the percentage 
value should be accepted in lieu of the other. 

To obtain the best idea of the opsonic content of the blood, estima- 
tions should be made not only with the concentrated serum, but also 
with dilutions, and I would recommend that 1 to 20 and 1 to 40 be 
accepted as standards; in that case the emulsion of organisms should 
be made rather dense so as to give values between 50 and 100 for 
concentrated normal blood. The best indeed would be if all opsonic 
workers were to accept a definite standard in this respect, as results 
would then be more directly comparable. 

Method. — The material necessary for an opsonic estimation is the 
following: 

The patient's serum., 

Normal control serum. 

Washed corpuscles. 

The bacterial emulsion. 



644 THE OPSONINS 

1. Preparation of the Patient's Serum. — A small amount of blood 
(about 6 or 8 drops) is collected from a puncture of the ear by means of 
a little pipette (Plate XXIV, Fig. a) and immediately transferred 
to a small glass tube having a diameter of about one-quarter of an inch 
(Plate XXIV, Fig. b), which is then tightly corked. The blood is 
allowed to clot, the coagulum separated from the walls of the tube, and 
the specimen centrifugalized (water or electrical centrifuge) until the 
corpuscles have been packed down and thus separated from the 
serum. 

Wright recommends the collection of the blood in special capsules 
(Plate XXIV, Fig. c), which are then sealed in a flame. He obtains 
the blood by puncturing the thumb near the root of the nail, after 
having previously allowed the arm to hang down and then applying 
some constriction hehind the distal joint (tape, rubber tubing). The 
puncture is made with a fine glass needle obtained by drawing 
out a piece of glass tubing in the flame of a small burner. When 
the bent capillary of the capsule is held to the exuding blood it enters 
by capillary attraction. On warming the body of the capsule the 
blood rises into it, when both ends are sealed. 

In my experience Wright's procedure offers no material advantages 
over the simpler method which I employ myself. The serum should 
not be older than twenty-four hours. 

2. Preparation of Normal Control Serum. — This is collected in the 
same manner as the patient's serum and separated from the corpuscles 
by centrifugatin. It is preferable to pool three or four normal 
sera, viz., to mix equal quantities from three or four individuals. If, 
however, the serum of one single person (of the experimenter, for 
example) has been thoroughly studied and always found normal, this 
single serum may suffice for all ordinary purposes. Women during 
menstruation, hard smokers, and individuals who are pale and below 
weight, even if otherwise healthy, should not be taken as control, nor 
included in a pool. Occasionally apparently normal individuals are 
also met with, who habitually have a higher opsonic content than nor- 
mal, and such must of course also be excluded. The process of 
digestion further tends to increase the opsonic content of the blood, 
so that it is advisable to take the blood of the patient and the pool 
approximately at the same time of the day. 

As with the patient's blood, the control serum should not be older 
than twenty-four hours; in my own work I use no blood that is older 
than twelve hours. 

3. Preparation of Washed Corpuscles.— The blood is most con- 
veniently collected from the ear and received in a tube containing 
either 1.5 per cent, of sodium citrate solution or 1.2 per cent, saline, 
containing 0.1 per cent, ammonium oxalate to prevent clotting. The 
amount will depend upon the number of specimens that are to be pre- 



PLATE XXIV. 




d 





Apparatus for Opsonic Work. 

(a) Pipette for collecting blood; (6) Tube to receive blood, for separation of 
serum; (c) Wright blood capsule; (d) Blood pipette charged with corpuscles, 
serum, and bacterial emulsion; (e) Same, in solid column, ready for incubation. 



TECHNIQUE 645 

pared; 1 c.c. of blood is sufficient for at least a dozen mounts. Small 
test-tubes of 5 c.c. capacity are very convenient. (The anticlotting 
fluid should be watched and the supply renewed when it becomes 
turbid.) Clots must be avoided and the specimen promptly discarded 
if the slightest coagulum has formed. This will rarely occur with 
reasonably fresh anticlotting fluid. The tubes are centrifugalized 
until the corpuscles have been well packed down and an opaque little 
film (of leukocytes) can be made out on top of the red cells. The 
supernatant fluid is then pipetted off and replaced with 1.2 per cent, 
saline; the washing is repeated once more and after the corpuscles 
have been again packed down the fluid is carefully withdrawn (the 
last traces with a capillary pipette). Wright then uses the superficial 
layer of corpuscles only,, as this is especially rich in leukocytes (the 
leukocytic cream). In my laboratory we shake up the cells thoroughly 
and find that we obtain a sufficient number of leukocytes in this way 
also. 

The washed corpuscles should not be kept longer than five or six 
hours. 

4. Preparation of the Bacterial Emulsions.— Perfectly uniform bac- 
terial emulsions cannot be secured as a matter of routine ; some clumps, 
if only of a few organisms, are practically unavoidable. For this 
reason I prefer the percentage index to the bacillary index, as it is 
not subject to errors arising from this source. 

With some organisms, an emulsion of a fair degree of uniformity 
is more readily obtained than with others; with the tubercle bacillus 
especially it is very difficult. 

Emulsions of Cocci. — Staphylococci and streptococci may be grown 
on plain agar, while gonococci, pneumococci, and meningococci are 
cultivated on blood agar or hydrocele agar. Small tubes, such as the 
one pictured on Plate XXIV, are charged with a little saline solution 
(0.85 to 1.2 per cent.). A bit of the culture is removed with a plati- 
num loop and very gently rubbed against the wall of the tube, at the 
surface of the liquid ; this must be done with a light hand, and slowly. 
When the fluid has become turbid it is centrifugalized for a few min- 
utes so as to remove clumps as far as possible and to acquire the 
proper degree of thickness. This point can only be learned by expe- 
rience ; trial tubes (see below) should be filled and specimens mounted 
from emulsions, showing various degrees of turbidity. Wright 
obtains the best result if four or five cocci are found pro cell, while 
with the percentage method I aim at a thinner emulsion, viz., one 
furnishing about 50 per cent, of phagocyting cells. Small glass 
capsules may be prepared containing emulsions of barium sulphate 
of varying degrees of turbidity, and corresponding to standard emul- 
sions of the various organisms; these are convenient in determining 
how far the centrifugation is to be carried. 



646 THE OPSONINS 

It has been recommended that the cultures should always be fresh 
and not more than twenty-four hours old. This is not necessary 
with all organisms. Knorr has shown that the same degree of pha- 
gocytosis is obtained with cultures of the staphylococcus more than 
a month old, as with young cultures, and in my laboratory we have 
worked successfully with one and the same emulsion preserved with 
a few drops of chloroform for a couple of months. 

Emulsions of Colon and Typhoid Bacilli. — Wright recommends the 
use of cultures only four hours old. With older cultures the spheru- 
lation of the organisms is such that approximate results only can be 
obtained. The percentage method with these organisms is certainly 
far superior to Wright's method. The emulsions are prepared in the 
same way as directed for the cocci. 

Emulsions of the Tubercle Bacillus. — Cole has obtained the best 
results by starting with living cultures of the tubercle bacillus on 
glycerin agar which are killed by exposure to sunlight for twenty- 
four hours. Some of the material is then scraped off, ground up in 
an agar mortar with 1.5 per cent, saline, and centrifugalized to remove 
clumps. If contamination is guarded against the supernatant emul- 
sion can be used for at least a month. 

I have not had an opportunity to use emulsions prepared in this 
manner, and am familiar only with material prepared from dried and 
ground-up dead bacilli. A small amount of these is placed in an agar 
mortar and ground up with 1.5 per cent, saline solution, which is 
slowly added drop by drop. The emulsion is centrifugalized free 
from coarse clumps, but always contains smaller ones which are 
practically impossible to remove. I have worked with extracted and 
non-extracted bacilli, with 0.1 and 1.5 per cent, saline, with heated 
and unheated bacilli, but have not yet seen an emulsion of the tubercle 
bacillus that was free from clumps. 

Wright recommends that the emulsion should be of such thick- 
ness that one or two organisms are found on an average for 
each cell, while I aim at approximately 50 per cent, of phagocyting 
cells. 

5. Charging the Pipette. — Having prepared the patient's serum, 
normal control serum, washed corpuscles, and bacterial emulsion, 
these tubes are conveniently placed in a dishful of sand covered with 
a piece of white filter paper, perforated to receive the tubes and 
marked accordingly. 

Mixing pipettes are prepared from glass tubing having an inside 
diameter of approximately 6 mm. (J inch). To this end pieces of 
tubing are cut, measuring about 15 cm. (6 inches) in length, and 
drawn out in the flame of a Bunsen burner, so that capillary stems 
are obtained about 15 to 18 cm. (6 to 7 inches) long, with an approxi- 
mate diameter of 1 mm., or a trifle less. The ends are cut off square 



TECHNIQ UE 647 

with a fine file. The tubes are marked about } to 1 inch from the 
ends with a glass pencil and provided before use with a rubber nipple 
(ordinary medicine-dropper nipples) . (See Plate XXIV. ) One volume 
of corpuscles is then drawn up to the mark, followed by one volume 
of serum and one of bacterial emulsion, the three portions being 
separated from one another by little bubbles of air. The contents of 
the tube are next blown out upon a slide, well mixed, drawn up and 
blown out repeatedly, and finally drawn up in a solid column, holding 
the pipette almost vertically to avoid bubbles of air. The end of the 
capillary stem is sealed in the flame of a Bunsen burner or an alcohol 
lamp and the tube incubated at 37° C. for fifteen minutes. 

6. The Slide Mount and Staining. — After incubation the end of 
the tube is pinched off, a large drop mounted upon a clean slide, 
stirred with the end of the tube, and a spread made with a second 
slide as in ordinary blood work, only a little thicker and using no 
force whatsoever. (See Fig. 17.) After drying in the air the specimens 
(excepting tubercle mounts) are stained without previous fixation, 
either with a 1 per cent, aqueous solution of methylene blue or 
with some polychrome dye like Jenner's, Hastings', Giemsa's, etc. I 
find the aqueous methylene blue especially convenient, as it largely 
removes the hemoglobin from the red cells; thicker specimens can 
thus be prepared and there will consequently be more leukocytes. If 
the leukocytic cream is used, thin preparations can be made and 
stained with polychrome dyes; the resulting pictures are very 
pretty. 

Tubercle specimens are fixed by immersion for ten minutes in a 
saturated aqueous solution of mercuric chloride. They are then 
washed off in water, stained with steaming carbol-fuchsin solution 
(Czaplewsky's formula, p. 345, foot note 5), washed in water, decolor- 
ized in 2 per cent, sulphuric acid, washed, immersed for a few seconds 
in 0.1 per cent, sodium carbonate solution, washed again, counter- 
stained for one minute with 1 per cent, aqueous methylene blue, 
washed once more, and set up to dry. 

7. Counting. — To obtain the bacillary index a series of 50 or more 
polynuclear leukocytes are examined and the number of organisms 
in each noted; the average for one represents the phagocytic index. 

To obtain the percentage index it is only necessary to note the 
number of phagocyting cells in the series and to work out the percent- 
age. The opsonic index in either case is calculated by dividing the 
patient's value by the normal control, as already described. 

To obtain reliable counts much practice is necessary, and every one 
will have to work out his own personal equation in deciding what 
organisms are to be counted in or out, when lying in the margin of the 
cell, whether a certain cell is to be excluded because it contains too 
many organisms to be counted, how many negative cells are to be 



648 THE OPSONINS 

thrown out to balance an eliminated positive cell, etc. For this 
reason the counts between two individuals will often vary considerably, 
unless a very large number of cells has been gone over, while for the 
same person comparative counts will agree much more closely. 

Literature. — Wright and Douglas, Proc. Royal Soc, 1903, vol. lxii, p. 357; 
ibid., 1904, vol. lxxiii, p. 128; ibid., p. 135; ibid., p. 147. Wright and Read, 
ibid., 1906, vol. lxxvii, p. 194, and ibid., p. 211. Hektoen and Rudiger, Journ. 
of Infect. Diseases, 1905, vol. ii, p. 128. Hektoen, Phagocytosis and Opsonins, 
Journ. Amer. Med. Assoc, May 12, 1906. Simon and Lamar, Johns Hopkins 
Hospital Bull., 1906,' vol. xvii, p. 27. Simon, Lamar, and Bispham, Journ. 
Exper. Med., 1906, vol. viii, p. 651. Simon, Journ. Amer. Med. Assoc, Jan. 12, 
1907, p. 138. Potter, Ditman, and Bradley, ibid., Nov. 24 and Dec. 1, 1906. 
Knorr, ibid., April 13, 1907, p. 1256. Ross, "The Opsonic Theory and its Appli- 
cation to Medicine and Surgery," Brit. Med. Journ., 1906. Bulloch, London 
Practitioner, Dec, 1905. Wright, Lancet, Dec. 2 and Dec. 9, 1905. Crace- 
Calvert, "Opsonins and the Opsonic Index and their Practical Value in the 
Treatment of Disease," Lancet, Feb. 2, 1907. 



APPENDIX. 



A. 
PREPARATION OF CULTURE MEDIA. 

Nutrient Bouillon. — Dissolve 6 to 8 grams of Liebig's beef extract 
together with 5 grams of sodium chloride and 10 grams of Witte peptone 
in about 100 c.c. of water by the aid of heat, stirring with a glass rod. 
Render the solution faintly alkaline to litmus (red paper should turn 
faintly blue, while the blue paper remains unchanged) by adding a 
fairly concentrated solution of sodium carbonate drop by drop. Or 
titrate 10 c.c. with T ^ normal alkali, using phenolphthalein as an indi- 
cator, to the point of the first pink which persists; estimate the cor- 
responding amount of normal alkali which must accordingly be 
added to the remaining bulk of the fluid; add this and dilute to 
1000 c.c. 

Example. — 10 c.c. required the addition of 10 c.c. of ~ normal 
alkali. There remain 90 c.c. of bouillon; for each 10 c.c. in this, viz., 
9, it is necessary to add 10 c.c. ~ alkali; so in this case 90 c.c. Instead 
of using so much of the -^ solution it is convenient to use 9 c.c. of 
the full-strength normal solution. This, however, is not necessary; 
the T \ normal, in the amount mentioned, can be used, if it only is 
available. 

If by any chance too much alkali has been added, use very dilute 
hydrochloric acid to return to the neutral point. 

In any event test the final reaction with litmus paper and see to 
it that the reaction is slightly but distinctly alkaline while blue litmus 
paper remains unchanged. Then filter into a liter flask, plug the 
mouth with cotton, and sterilize for one hour in the steam sterilizer. 
After that tubes are filled to the desired height (1J to 2 inches) and 
again sterilized. 

Glucose Bouillon. — This is nutrient bouillon to which 1 to 2 per cent, 
of glucose has been added. 

Lactose Bouillon. — Nutrient bouillon, containing 1 to 2 per cent, of 
lactose. 

Other carbohydrate bouillons contain corresponding amounts of 
material. 



( 



650 APPENDIX 

Nutrient Gelatin. — 6 to 8 grams of Liebig's beef extract, 5 grams of 
sodium chloride, and 10 grams of Witte peptone are dissolved in a 
liter of water, as in the preparation of nutrient bouillon. To this 
solution 100 to 150 grams of gelatin are added, the latter broken up 
into small pieces. The mixture is boiled in an agate saucepan, 
stirring frequently so as not to burn the gelatin at the bottom. It is 
then neutralized as described above (preparation of nutrient bouillon), 
and clarified by the addition of the white of an egg beaten up in 
50 c.c. of water. Before this is added the solution should be allowed 
to cool to 60° C. After this the boiling is continued for fifteen minutes, 
allowance being made for evaporation by the addition of a little water 
from time to time. The solution is then filtered. To this end no 
hot-water funnel or other artificial contrivance is necessary. The 
essential requisite is that the gelatin is in solution and has been 
actually boiling. The filter is wetted thoroughly before; if the first 
4 c.c. should pass through turbid they are passed back. If the fil- 
tration should cease, the material in the funnel must be further 
boiled and the filtration continued thereafter. 

The filtrate is received in a flask, plugged with cotton, and ster- 
ilized on three consecutive days in the Arnold sterilizer for fifteen to 
twenty minutes daily. Tubes, however, can be charged on the first 
day and the sterilization carried on in these. 

Nutrient Agar. — This consists of nutrient bouillon, containg 1 to 1.5 
per cent, of agar. The agar (10 to 15 grams) is cut into very small 
pieces and placed for twenty-four to forty-eight hours in 600 c.c. of 
water containing the 5 grams of salt required for the liter of bouillon. 
In the mean time the 6 to 8 grams of Liebig's beef extract and 10 
grams of peptone are dissolved in 400 c.c. of water, neutralized as 
described (see Nutrient Bouillon), and sterilized. After soaking as 
indicated, the agar-salt mixture and the neutralized beef-peptone solu- 
tion are poured together in an agate saucepan and the depth of the 
liquid measured; 300 c.c. of water are then added to allow for 
evaporation during the two hours and a half of active boiling which 
must follow. During this process the liquid must not fall below its 
original bulk. The white of an egg beaten up in 50 c.c. of water is 
then added (the liquid should be previously allowed to cool to 60° C. 
by setting the pan in a vessel with cold water), after which the boiling 
is continued actively for half an hour longer, when the agar is filtered 
through a previously prepared filter which has been well wetted. 
If the agar is well in solution the liter will pass through in little more 
then half an hour. If filtration should stop, the material must be 
boiled again and a new filter prepared. The agar can be filtered 
into tubes the same day or kept in a plugged flask ; in either case it 
must be sterilized for three consecutive days in the steam sterilizer 
for fifteen to twenty minutes daily. 

If agar slants are to be prepared, care must be taken not to fill the 



PREPARATION OF CULTURE MEDIA 651 

tubes too high. After their final sterilization they are laid down, 
slightly elevated at the open end, so that the agar forms a long 
slant; in this position they remain for some hours (over night). 

Glycerin Agar. — This is nutrient agar containing 6 to 8 per cent, of 
glycerin. This is added after filtration and before sterilization. 

Glucose Agar. — This is nutrient agar containing 1 to 2 per cent, of 
glucose. The glucose is conveniently dissolved in the beef extract- 
peptone portion. 

Other carbohydrate agars contain corresponding amounts of 
material. 

Litmus Agar. — This is ordinary agar which has been colored by 
the addition of a 5 per cent, solution of purified litmus; the agar 
should show a bluish color. 

Litmus- Carbohydrate Agar. — Litmus agar containing 1 per cent, of 
one of the various carbohydrates — dextrose, lactose, mannite, etc. 

Hydrocele Agar (Cushing). — The fluid (hydrocele or ascitic) is 
obtained sterile, the locality of puncture being carefully sterilized by 
modern surgical methods, the sterile trocar covered at its external 
end with sterile gauze, so as not to be infected by the operator's hand, 
and the fluid collected in sterile flasks, the sterile stoppers being then 
replaced. When collected in this way it rarely becomes contami- 
nated and may often be kept for months before using. This fluid 
is mixed with ordinary nutrient agar. A number of common agar 
slants are placed in the autoclave for five minutes. This liquefies 
the agar and at the same time thoroughly sterilizes the tubes and 
cotton stoppers. The slants are then put in a water bath at 55° C, 
so as not to coagulate the albumin when mixed with the agar. 
The stopper having been removed from a small flask of hydrocele 
fluid, the top of the flask is flamed and the albuminous fluid then 
poured into an agar tube (the top of which has also been flamed) in 
the proportion of a little more than 1 to 2. It is well to have 
as much of the hydrocele fluid as the future solidity of the medium 
will allow. Ordinary agar will allow not quite equal parts of the 
two. The stopper is then returned to the agar tube, which is 
immediately slanted. On these slants gonococci grow most abun- 
dantly in or near the liquid which is squeezed out of the medium 
and collects at the bottom of the tube. Some cultures will main- 
tain a vigorous growth after numerous transplantations, while others 
again grow only two or three times, or indeed once only. 

Blood Agar. — Agar tubes are melted, as just described, and then 
placed in a water bath at 50° C. To each tube approximately one- 
half of a cubic centimeter of human blood is added. Agar and blood 
are well mixed and the tubes immediately slanted. Before use they 
should be incubated for twenty-four hours to see that they are sterile. 
The necessary blood is obtained by aspirating a vein with a sterile 
syringe, containing a little 1 per cent, sodium citrate to prevent 



652 APPENDIX 

coagulation, or it may be collected in a sterile glass pipette Lorn the 
ear under antiseptic' precautions. 

Neutral Red Agar. — Agar 2 per cent., grape sugar 0.3 per cent., 
neutral red solution 1 c.c. (saturated watery solution of Ehrlich's 
neutral red). Mix; sterilize. 

Dunham's Solution. — This is common nutrient bouillon without the 
addition of Liebig's beef extract. Its reaction is neutral or slightly 
alkaline per se, and need hence not be corrected. The solution is 
filtered, tubes filled and sterilized, as in the case of bouillon. 

Litmus Milk. — Fresh milk which has been freed from cream, as 
far as possible, is treated with tincture of litmus until it presents a 
distinct blue color. Tubes are filled with this and sterilized on two 
successive days for an hour at a time. 

Litmus Whey.— To 500 c.c. of milk add 10 to 12 c.c. £ solution HC1 
to precipitate the casein. Neutralize with soda solution. Boil one to 
two hours. Let the precipitate fall to the bottom. Take 100 c.c. cf 
fluid and add 5 c.c. litmus solution. Place in tubes; sterilize for 
from two to three hours at 100° C. 

Potato Slant. — Large potatoes are selected. They are thoroughly 
scrubbed in running water and cylinders forced out with a large cork 
borer. They are cut square at the ends and then obliquely into two 
parts. The resultant wedges are kept over night in running water 
and the next day are placed in sterile tubes. The potato tubes are 
steamed for one hour. 

Loeffler's Blood Serum. — 3 parts of ox-blood serum are mixed with 
1 part of nutrient bouillon containing 1 per cent, of glucose. Tubes 
are filled with this mixture and coagulated in a slanting position 
in the drying oven at a temperature a little above 90° C. It is impor- 
tant to raise the temperature to this point quite gradually. Here they 
remain until the slants are quite firm, after which they are sterilized 
in the steam sterilizer at 100° C. for fifteen minutes at a time, on three 
consecutive days. 

The blood necessary for the preparation of the medium is pro- 
cured at a slaughter-house. Care should be taken that it flows 
directly from the cut vessel into a suitable receptacle, which has been 
previously sterilized. Museum jars are convenient for this purpose. 
After coagulation has set in the coagulum is carefully separated from 
the walls of the vessel with a sterile glass rod and the blood kept in 
a cool place (ice-box). The serum which separates out is pipetted 
off wi h sterile pipette and placed in sterilized and plugged cylinders 
or test-tubes until required. 

Two gallons of blood will approximately yield from 500 to 700 c.c. 
of serum. 

Hiss' Serum-water Media. — The serum water is composed of beef 
serum 1 part and distilled water 2 or 3 parts. To this 1 per cent, of 
a 5 per cent, solution of highly purified litmus is added. The medium 



PREPARATION OF CULTURE MEDIA 653 

is heated for a few moments to 100° C, when 1 per cent, of either 
dextrose, lactose, maltose, saccharose, raffinose, dextrin, glycogen,, 
inulin, mannite, or dulcite is added. Tubes are then filtered and 
sterilized on three consecutive days by steam at 100° C. for fifteen 
minutes at a time. 

The Drigalski-Conradi Medium. 1. Agar Preparation. — To 3 
pounds of finely cut beef add 2 liters of water. Allow it to stand 
till next day. The expressed meat juice is boiled for one hour and 
filtered. Add 20 grms. of Witte peptone, 20 grms. nutrose, 10 grms. 
NaCl; boil one hour, now add 70 grms. bar agar, then boil three 
hours (or one hour in autoclave), render slightly alkaline (indicator 
litmus paper). Filter; boil half an hour. 

2. Litmus Solution. — Litmus solution (Kubel and Tieman) 260 
c.c, boil for ten minutes; add milk sugar (chemically pure) 30 grms. 
Boil fifteen minutes. 

3. Add the hot litmus-milk-sugar solution to the liquid agar solu- 
tion cooled to 60° C. Shake well. Render it again faintly alka- 
line. The color of the froth is a good indicator. Add then 2 c.c. 
of a hot sterile solution of 10 per cent, water-free soda; further add 
20 c.c. of a freshly prepared solution of 0.1 grm. crystal-violet B. 
(Hochst) in 100 c.c. of warm water (distilled). 

One has now a meat-water peptone-nutrose agar with 13 per 
cent, litmus and 0.01 per mille crystal violet. This can be poured 
directly into plates and the remainder kept in 200 c.c. flasks. 

The Malachite-green Enriching Method of Lentz. — The proper prepa- 
ration to use is malachite green (crystal) (Hochst); dilution 1 to 
22,000. Preparation: 3 pounds of lean beef, finely divided, are 
macerated with 2 liters of water for sixteen hours. The extract is 
expressed, boiled for half an hour, filtered, then 3 per cent, agar 
added and boiled for three hours; then add 1 per cent, peptone, 
0.5 per cent. NaCl, and 1 per cent, nutrose (this may be omitted). 
The mixture is brought to the litmus-neutral point by soda solution, 
boiled one hour, and filtered through linen. The reaction of the 
finished agar is sometimes distinctly acid. It is filtered into small 
flasks of 100 to 200 c.c. 

Before the addition of the malachite green the hot agar is tested 
with neutral litmus paper and so far alkalinized with sterile soda solu- 
tion until the strip is distinctly red-violet. This reaction point cor- 
responds in agar, without nutrose, to an alkalescent degree of 1.8 . 
per cent, normal soda below the phenolphthalein-neutral point; if the 
agar contains nutrose, which remains neutral toward litmus, then the 
alkaline reaction corresponds to 3.5 per cent, normal soda solution 
below the phenolphthalein point. 

To 100 c.c. of the hot agar 1 c.c. of a 1 to 220 solution of malachite 
green (the solution is stable for ten days) is added; the agar thus 
contains 1 in 22,000. With this concentration *of malachite green 



654 APPENDIX 

(crystal) the growth of the usual kinds of B. coli, as well as many 
alkali-forming organisms, is greatly diminshed and practically pre- 
vented. 

The B. typhosus growth is also diminished, but only so far that 
after twenty-four hours the colonies can be recognized with the 
naked eye; they are then the size of a particle of sand, while, after a 
longer period in the incubator, in two to four days, larger, stronger 
colonies appear which color the agar yellow. 

The finished agar is poured at once into Petri dishes in layers 
2 mm. thick. 1 



B. 

OUTLINE OF A COURSE IN CLINICAL LABORATORY 

METHODS. 

In response to numerous requests I have arranged below a program 
of practical instruction in the clinical laboratory. This is based 
essentially upon the work done in my private courses which I have 
conducted for a number of years for postgraduates, and may have to 
be modified more or less to meet special requirements. The students 
who come to me for instruction are mostly general practitioners, and 
for their special needs this program has been arranged. The practi- 
cal work is supplemented by lectures of a more or less formal and 
comprehensive character, as indicated below. The exercises have 
been collectively arranged so as to correspond to the general topics: 
Blood, Gastric Juice, Feces, Sputum, etc. This routine may, how- 
ever, be interrupted, as special material becomes available, which 
cannot be advantageously preserved and should hence be examined 
at once. 

1. Lectures. A. Blood. — 1. General technique: the morphology of 
the blood, studied in the whet specimen; classification of the leuko- 
cytes, as seen in the whet specimen ; percentage values ; general account 
of variations in disease. 

2. The chemistry of the aniline dyes : structure; classification ; forma- 
tion of neutral dyes; meaning of the terms neutrophilic, basophilic, 
oxyphilic, monochromatophilic, polychromatophilic, etc. Classifica- 

1 The plates are allowed to remain open until all the steam has evaporated and 
the agar is stiff. It is essential that the surface of the plates should be quite 
dry and firm. Contamination by air organisms does not occur on account of 
the aniline dye present in the culture media. 



A COURSE IN CLINICAL LABORATORY METHODS 655 

tion of the leukocytes upon the basis of their behavior toward aniline 
dyes. 

3. Preparation of stains and methods of. staining: Jenner's, 
Giemsa's, Hastings', Goldhorn's, Ehrlich's stain. 

4. Leukocytosis : significance of variations in the absolute number 
and of the relative percentages; neutrophilic hyperleukocytosis and 
the septic factor; eosinophilia ; lymphocytosis; large mononucleosis; 
increase of mast-cells; leucopenia. 

5. Origin and interrelation of the various leukocytes: myelocytes; 
metamyelocytes; meaning of the polymorphism of the nucleus and 
actual polynucleosis. Arneth's findings. 

6. Leukemia. 

7. The red corpuscles: poikilocytosis, anisocytosis ; staining prop- 
erties; granular degeneration; polychromatophilia; origin of the 
normocytes; normoblasts and megaloblasts ; relation to leukocytes. 

8. 'Clinical variations in the number of the red cells and the 
amount of hemoglobin ; color index. 

9. Pernicious anemia. 

10. The hemocytometer and hemoglobinometer. 

11. The bacteriological examination of the blood; technique and 
ciinical indications; typhoid fever, pneumonia, pyogenic septicemia. 

12. The serology of the blood: meaning of the term antigen, anti- 
body, immunization, vaccination. Classification of the antibodies. 
Discussion of the formation of antitoxins, cytolysins, bacteriolysins, 
hemolysins, precipitins, coagulins, agglutinins, and antiferments. 

13. The opsonins. 

14. Malaria: life cycle of the malarial organism; asexual and sexual 
reproduction; methods of staining and general technique. Try- 
panosomiasis; relapsing fever; Kala-azar; filariasis. 

B. Gastric Juice. — 1. The gastric juice; secretion; chemical com- 
position; test meals and the rationale of their employment; free 
hydrochloric acid and combined hydrochloric acid; euchlorhydria, 
hypochlorhydria ; anachlorhydria and their clinical significance. 

2. Analysis of the acid factors of the gastric juice: meaning 
of the terms normal solution, decinormal solution, indicators; 
technique. 

3. The organic acids of the stomach contents: their origin and 
clinical significance; analytical methods. 

4. The gastric ferments: their specific action; clinical significance 
of quantitative variations; analytical methods. 

5. The present status of our knowledge of proteolytic digestion: 
concept of the terms albumose, peptone, polypeptid, etc.; proteid 
synthesis. 

6. The microscopic constituents of the stomach contents: alimen- 
tary detritus, yeast, sarcina?, Boas-Oppler bacillus, protozoa, tumor 
particles; technique. 



656 APPENDIX 

7. The significance of the presence of blood in the stomach con- 
tents: tests for occult bleeding. 

C. The Feces. — 1. The chemistry and microscopy of normal feces; 
technique. 

2. Animal parasites occurring in the intestinal tract and their 
clinical significance. Methods of examination. 

3. The bacteriology of the feces. 

4. The significance of the presence of blood in the feces: occult 
bleeding; technique. 

D. The Sputum. — 1. General account of information to be derived 
from a microscopic study of the sputum, with special reference to 
tuberculosis, pneumonia, influenza, asthma, bronchiectasis, abscess 
of the lung, gangrene of the lung; general technique. 

2. The tubercle bacillus and related organisms; special methods. 

E. Exudates. — 1 . The bacteriology of tonsillar exudates : diphtheria, 
tonsillitis, stomatitis, Vincent's angina; pharyngomycosis leptothricia; 
technique. 

2. The pus in gonorrhea: the gonococcus; the pseudogonococci; 
pus eosinophilia. 

3. Syphilis and the Spirochete pallida. 

4. The cytological and bacteriological study of pleural and peri- 
toneal exudates. 

5. The cytological and bacteriological study of cerebrospinal fluid. 

F. The Urine. — 1. General chemical study of the urine and dis- 
cussion of its relative importance in the diagnosis of various patho- 
logical conditions. 

2. The urinary constituents: chlorides, phosphates, sulphates. 

3. Urea and nitrogenous metabolism. 

4. Metabolic anomalies: lithuria, oxaluria, cystinuria, diaminuria, 
alkaptonuria. 

5. Metabolic anomalies: diabetes and carbohydrate metabolism. 

6. Albuminuria and its clinical significance. 

7. The various pigments and chromogens which may occur in the 
urine and their clinical significance : indicanuria, melanuria, the diazo 
reaction, bilirubinuria, urobilinuria. 

8. The microscopic study of the urine: technique; the non- 
organized components of urinary sediments; the significance of such 
deposits. 

9. The microscopic study of the urine: the organized components 
of urinary sediments — tube casts, pus, blood, epithelium. 

10. The bacteriological study of the urine: renal tuberculosis and 
its diagnosis; the typhoid bacillus, the colon bacillus; technique. 

G. The Milk. — Milk analysis and its indications. 

II. Laboratory Exercises. Blood. — In all microscopic exercises an 
examination with the low power (B. and L. §; Leitz 3; Spencer 16) 



A COURSE IN CLINICAL LABORATORY METHODS 657 

should precede the use of the higher powers. The instructor should 
insist that the s udent draws what he sees and keeps careful notes of 
his work. 

Exercise I. Having cleansed cover-glasses and slides (p. 120) mount 
a normal wet specimen (p. 121). Note (a) the form and size of the 
red cells, rouleaux formation and crenation (pp. 51 to 53); (b) the 
different kinds of leukocytes, viz., non-granular mononuclear forms 
and granular polynuclear forms (pp. 69 to 70); (c) the plaques 
(p. 118); (d) the hemokonia (p. 120). Note the ameboid move- 
ments of the granular leukocytes and the changes in outline of the 
mononuclear forms. 

Exercise II. Repeat lesson I. Then prepare a dry normal speci- 
men; stain with eosinate of methylene-blue solution (p. 129). Study 
the appearance of the red cells, the various types of leukocytes (pp. 
70 to 80), and the plaques. Make a differential leukocyte count of 
at least 300 cells (p. 143) ; make an Arneth count of 100 neutrophiles 

Exercise III. Make a differential count of blood from cases of 
pneumonia, active appendicitis, wound infection, and note the septic 
factor (p. 90). Make Arneth counts from the same cases. Study 
the iodophilia in these cases (p. 137). 

Exercise IV. Make differential counts in cases of trichinosis, hook- 
worm infection, acute gonorrhea, bronchial asthma, and note the 
extent of eosinophilia (p. 102). 

Exercise V. Make differential counts of typhoid blood from different 
stages of the disease. Note the early tendency to lymphocytosis and 
large mononucleosis. Make an Arneth count and note that although 
the number of neutrophiles is not increased there is evidence of 
marked changes in the relative percentages of the different types (p. 
101). Ascertain in suitable cases how early this disproportion of the 
neutrophilic blood picture occurs. 

Exercise VI. Study specimens of blood from cases of whooping- 
cough, mumps, rickets, congenital syphilis, and note the degree of 
lymphocytosis (p. 109). 

Exercise VII. Study the large mononuclears in cases of malaria of 
short and long standing; note the tendency to an increase in cases of 
the latter kind (p. 113). 

■ Exercise VIII. Study smears from the red bone-marrow (human), 
stained with eosinate of methylene blue, and note the presence of 
myelocytes (p. 80) and nucleated red cells (p. 65) ; draw the various 
types of cells ; make similar smears from a lymph gland (cat) and the 
spleen; note the character of the mononuclear elements and draw 
them. 

Exercise IX. Study the blood from a case of myelocytic leukemia (p. 
116) ; make a differential count, introducing into the eye-piece a small 
paper diaphragm with a small rectangular aperture, so that only a 
42 



658 APPENDIX 

half-dozen cells, or but a few more, appear in the field at one time. 
Make an Arneth count of the neutrophilic elements. Note the mye- 
locytes, metamyelocytes, monokaryolobic leukocytes, polykaryolobic 
leukocytes; draw. Contrast this type of hyperleukocytosis with 
that seen in pyogenic infections. Study the staining qualities of the 
eosinophilic myelocytes and contrast with what is seen in the adult 
forms; draw. Note the increase of the mast-cells; draw. Look for 
evidences of nuclear division in the myelocytes. Study the variations 
in the size of the different leukocytes. Note the presence of isolated 
large lymphocytes. 

Study the nucleated red cells; look for mitoses, nuclei undergoing 
karyolysis, free nuclei; note the polychromatophilic protoplasm of 
some of the nucleated red cells. 

Exercise X. Study the blood from cases of acute and chronic lym- 
phocytic leukemia (p. 112). 

Exercise XL Study the red cells in stained specimens of blood from 
a case of chlorosis, a severe secondary anemia the result of a pyo- 
genic infection, chronic lead intoxication, carcinomatous cachexia, 
pernicious anemia. Compare carefully the size of the cells, their 
form and their apparent amount of hemoglobin; draw. Look for 
polychromatophilic red cells, granular degeneration, and nucleated red 
cells. Contrast megaloblasts and normoblasts in the blood of per- 
nicious anemia and myelocytic leukemia; note especially the size, form, 
and structure of the nucleus; note small and large (young and old) 
megaloblasts and young and old normoblasts; nuclei in karyorhexis; 
draw (pp. 51 to 53, p. 60, and pp. 61 to 69). 

Exercise XII. Make a red and white count in a normal individual, 
using the hemocytometer (pp. 137 to 141). Make a dry normal 
mount, stain with eosinate of methylene blue, wash with water; 
examine the specimen while still wet, with a low power (B. and L. 
J; Leitz 3; Spencer 16), and note the number of blue specks (leuko- 
cytes, in the thinner and thicker parts of the spread; note that this is a 
normal case. Look at mounts from different infections (smeared with 
a little oil on the surface to obtain satisfactory refraction), and gauge 
the number of leukocytes by comparing with the normal. Practise 
this in concrete cases, controlling your findings with the hemocy- 
tometer. Make a differential count with the low power and practise 
this thoroughly. 

Exercise XIII. Estimate the amount of hemoglobin with different 
hemoglobinometers (Fleischl, Dare, Sahli, Talquist) (pp. 147 to 154). 

Exercise XIV. Determine the color index in a given case (p. 53). 

Exercise XV. Prepare eosinate of methylene blue (p. 129) and some 
modification of the Romanowsky stain (p. 132). 

Exercise XVI. Stain specimens with Hastings', Goldhorn's or 
Giemsa's stain, and also with Ehrlich's stain (the latter, after fixing 
by heat) (pp. 130, 132, 136, and 137). 



A COURSE IN CLINICAL LABORATORY METHODS 659 

Exercise XVII. Study specimens of malarial blood, stained with a 
Roman owsky mixture, and also with eosinate of methylene blue. 
Work out the different stages of development of tjie malarial organism 
in the different types of fever. Study, if possible the fresh, unstained 
blood also (pp. 177 to 187). 

Exercise XVIII. Repeat lesson XVII, and examine also dehemo- 
globinized specimens, prepared according to Ruge's modification of 
Ross' method (p. 177). 

Exercise XIX. Study the distinguishing characterstics of Anopheles, 
Culex, and Stegomyia mosquitoes ; examine their eggs, larvse, and pubse. 

Exercise XX. Study preparations showing the development of the 
malarial organism in the body of the mosquito. 

Exercise XXI. Study preparations of trypanosomes, Leishmania- 
Donovani, recurrens spirochsetse, and filarial (pp. 187, 169, 190, and 
191). 

Exercise XXII. Make the agglutination test in a well-advanced 
case of typhoid fever (pp. 161 and 164). 

Exercise XXIII. Make a bacteriological examination of the blood 
in a case of typhoid fever (pp. 158 and 159). 

Exercise XXIV. Determine the opsonic index in a given case (a) 
for the Staphylococcus aureus, (6) for the tubercle bacillus (p. 646). 

B. Gastric Contents. — Prepare an artificial "gastric juice" according 
to the following formula: 0.3 per cent, solution of hydrochloric acid, 
500 c.c.; pepsin powder, 2 grms.; bread, 40 grms. 

Place this in the incubator for thirty minutes, filter, and then examine 
as follows: 

Exercise I. Test the reaction with litmus paper; test for free acid 
with Congo red (p. 219) ; test for free HC1 with dimethyl-amino-azo- 
benzol (p. 219), phloroglucin vanillin (p. 220), and tropseolin (p. 221). 

Determine the acid factors: 

(a) The total acidity in 10 c.c, and express your results in terms 
of c.c. of j-q alkali solution for 100 c.c. of gastric juice (p. 222). 

(b) The alizarin acidity in a similar portion (free acids and acid 
salts); deduct this value from the total acidity; the result gives the 
acidity referable to combined HC1 (p. 222). 

(c) The free HC1 in a third portion of 10 c.c. (p. 222); b plus c 
gives the total amount of HC1; this deducted from a indicates the 
acidity due to acid salts. 

Apply the biuret test to 10 c.c. of stomach contents: render strongly 
alkaline with caustic alkali, and add a 2 per cent, solution of CuS0 4 
solution, drop by drop. 

Exercise II. Repeat exercise I and demonstrate further the pres- 
ence of pepsin (p. 229) and of rennin (p. 231). Estimate the amount 
of pepsin according to Metts' method (p. 229). 

Exercise III. Prepare an acid mixture as above, substituting lactic 
acid for the hydrochloric acid. Apply (a) Kelling's test, (b) Uriel- 



660 APPENDIX 

mann's test (after extracting with ether); estimate the amount of 
lactic acid according to Boas' briefer method (pp. 235 and 240). 

Exercise IV. Secure stomach contents from a case of carcinoma of 
the stomach, after Boas' test meal. Demonstrate the presence of 
lactic acid and estimate its amount; show the absence of free HC1; 
test for pepsin and rennin, viz., pepsinogen and renninogen. 

Examine the sediment of the contents, by mounting a drop on a 
slide and covering with a cover glass ; note the starch granules (stain 
with Lugol's solution) ; search for Boas-Oppler bacilli in a whet speci- 
men and in a smear stained with a 1 per cent, aqueous solution of 
methylene blue; so also for yeast cells and bacteria in general; look 
for pus corpuscles (neutrophilic leukocytes), red cells, and protozoa 
(p. 249). 

Exercise V. Examine the contents from exercise IV for occult 
blood (p. 260). 

Exercise VI. Procure stomach contents from a case of dilatation 
in the morning before food has been taken. Note the amount of 
fluid and of residual food material. Examine microscopically and 
chemically as above. If much HC1 is present sarcinse may be found ; 
if not, examine a droplet of an emulsion of sarcinse obtained from a 
culture; also make a smear and stain with methylene blue. 

Exercise VII. Prepare a T ^ solution of sodium hydrate and stand- 
ardize it against T \ oxalic acid (p. 216). 

C. Feces. — Exercise I. Procure some 'normal feces, stir up with 
normal salt solution to a thin mush; mount droplets, further diluted, 
on slides, and cover with cover-glasses. Note that the feces are largely 
composed of bacteria. Here and there muscle fibers may be found, 
more or less well preserved and all stained yellow. Look for fat 
globules and stain with a drop of Sudan III solution (fat-red) (p. 266) ; 
search also for starch (add a drop of Lugol's solution; starch colored 
blue) ; look for fatty acid needles ; draw. 

Make smears from the same stool and stain with methylene blue; 
note the great variety of bacteria; draw. 

Exercise II. Repeat exercise I with a diarrheal stool. Look for 
particles of mucus and pus. Examine wet specimens and smears, 
stained with methylene blue; draw. 

Exercise III. Provide stools containing ova of the hookworm, 
tapeworm, and Trichocephalus dispar; study and draw these; furnish 
eggs from other intestinal parasites; draw (pp. 295 to 316). 

Study the corresponding worms; draw. 

Exercise IV. Provide trichinous meat and study the corresponding 
parasite (p. 311); review exercise III. 

Exercise V. Study the Amoeba coli in suitable stools (vital staining 
with neutral red) ; so also the trichomonas; draw. Such stools cannot 
be adequately preserved, while most others can be kept in 1 per cent, 
carbolic acid (pp. 286 and 291). 



A COURSE IN CLINICAL LABORATORY METHODS 661 

Exercise VI. Provide a stool containing blood and pus; note 
especially the size of the pus cells; examine microscopically; demon- 
strate the presence of blood chemically (p. 260). 

D. Sputum. — In working with sputa treat all specimens as though 
they were tuberculous. 

Exercise I. Examine wet mounts of scrapings from the tongue and 
teeth; note the character of the epithelium, mucous corpuscles, and 
the large number and variety of microorganisms (among these spiro- 
chete) . Prepare smears on slides and stain with methylene blue; 
draw. 

Exercise II. Examine sputa from cases of acute and chronic bron- 
chitis, asthma, and chronic heart lesions as in I; note the epithelial 
elements (myelin, fat, and pigment granules), pus cells, red corpuscles, 
crystals. In stained specimens search for eosinophiles and note their 
number (pp. 334 to 337). 

Exercise III. Examine tuberculous sputa for the tubercle bacillus, 
according to Gabbett's method; examine for elastic tissue (procure 
material from an advanced case with cavity formation) (pp. 337 
and 345). 

Exercise IV. Examine pneumonic sputa ; note their physical charac- 
teristics, color, and odor; demonstrate the pneumococcus arid attempt 
its isolation. Stain the capsules according to Burger's method (p. 
348). 

E. Exudates. — Exercise I. Prepare smears from tonsillar deposits 
and stain with alkaline methylene-blue solution; note the pres- 
ence of staphylococci, streptococci, and diphtheria bacilli; examine 
cultures of the latter grown on blood serum; make smears and stain 
according to Neisser's method; note the polar bodies; draw (p. 205). 

Study specimens from Vincent's angina (spirilla and fusiform 
bacilli) (p. 204). 

Exercise II. Prepare smears of pus from an acute case of gonorrhea ; 
stain a specimen with eosinate of methylene blue; note (a) the gono- 
cocci in the pus cells (neutrophilic leukocytes); (b) the presence of 
eosinophilic leukocytes. Stain another specimen according to Gram. 
(p. 602). 

Obtain some gonorrheal threads from the urine; make smears of 
these and examine in the same manner; draw. 

Exercise III. Obtain smears of syphilitic serum from chancres, 
papules, and condylomata and stain according to Goldhorn's method; 
note the S. refringens and the pallida; contrast the two and draw 
(p. 606). 

Exercise IV. Procure a serous pleural exudate, determine its cyto- 
logical formula, and search for organisms (tubercle bacilli) according 
to Jousset's method. Also determine the specific gravity of the fluid 
and the amount of albumin (pp. 592 and 596). 

Contrast these various factors with a transudate. 



662 APPENDIX 

Exercise V. Examine cerebrospinal fluid in the same manner as in 
IV. 

Exercise VI. Examine by culture methods a specimen of an exu- 
date obtained at operation (peritonitis). 

F. The Urine. — Exercise I. Collect the urine of twenty-four hours 
from a normal individual, preserve with a teaspoonful of chloroform, 
which should be added to the first portion voided, shaking well after 
every addition. Note the color, odor, and transparency, its reaction 
to litmus, and specific gravity. Estimate the acidity according to 
Folin's method (pp. 360 to 373). 

Estimate the chlorides according to Volhard's method (p. 377). 

Exercise II. Estimate the urea (p. 411) and uric acid (p. 424). 

Exercise III. Examine microscopically the sediment of a dozen 
specimens of normal urine (p. 546); note the epithelial elements 
(especially in the urine of women), their number and form, crystals 
and mucous cylinders; draw. Let one specimen stand exposed to 
the air without a preservative for forty-eight hours and reexamine; 
note the development of bacteria and the abundant sediment; add 
acetic acid and note that the amorphous portion largely dissolves. 
How would you distinguish such a sediment from one due to 
amorphous urates? 

Exercise IV. Procure urine from a case of active nephritis. 
Examine for albumin, using the various tests described on pp. 461 to 
466. Estimate the amount in an Esbach tube (p. 467). Examine 
microscopically for tube casts (p. 570). 

Exercise V. Procure urine from a patient after a somewhat pro- 
longed ether anesthesia and examine as in IV. 

Exercise VI. Procure urine from different cases of nephritis and 
examine as in IV; contrast the findings in an arteriosclerotic case 
with those in an acute process, in association with typhoid fever 
or scarlet fever. Stain some of the sediment on the slide with a 
little eosin and note how the different kinds of casts are colored. 
Draw hyaline, granular, brown, epithelial, pus, blood, and waxy 
casts. 

Exercise VII. Add 10 grams of glucose to 500 c.c. of urine and 
examine qualitatively for sugar, using (a) Fehling's test, (b) Nylander's 
test, (c) the fermentation test, (d) the phenylhydrazin test (purify 
the osazone crystals in the latter case and determine their melting 
point (pp. 483 to 487). Estimate the quantity of sugar (a) by Fehling's 
method (p. 489), (b) with the polarimeter (p. 494). 

Exercise VIII. Repeat exercise VII with the urine from an active 
case of diabetes; test also for acetone directly; distil some of the urine 
and demonstrate acetone in -the distillate (Lieben's test, Legal's test, 
Gunning's test, etc., p. 529). Test for diacetic acid. Add some 
/3-oxybutyric acid to the urine, demonstrate its presence, and esti- 
mate the amount (p. 532). 



A COURSE IN CLINICAL LABORATORY METHODS 663 

Exercise IX. Procure urine from a typhoid patient, about the tenth 
day of the disease. Apply the diazo test (p. 524) and also the indi- 
can test (p. 503). Test normal urine in the same way and compare 
the colors. In doing the diazo reaction do not fail to practise the 
diluting method in the end and compare the color with the normal. 

Exercise X. Procure urine from a case of jaundice and apply the 
various tests for bile pigment (p. 512); contrast the results with a 
urine rich in urobilin; demonstrate the presence of the latter (p. 515). 

Exercise XL Procure urine from cases of cystitis and pyelitis and 
study these microscopically (pus corpuscles, red cells, epithelial cells). 
Examine for tubercle bacilli by Gabbett's method and inject a guinea- 
pig with sediment from a tuberculous case (autopsy after three weeks). 

Exercise XII. Show some of the rarer crystalline elements, such 
as cystin, leucin, tyrosin, fatty acids, xanthin, hippuric acid, etc., 
and review sediments in general thoroughly. 

G. Milk. — Exercise I. Determine the specific gravity of a specimen 
of milk. Estimate the amount, of fat (p. 637), of lactose (p. 637), 
and of proteids (p. 637). 

Examine a droplet of milk microscopically; note the fat globules 
and their behavior toward Sudan III. 

Exercise II. Examine colostrum as in exercise I. 



INDEX. 



Abortion, vaginal discharge in, 631 
Acetic acid, 241 

fermentation, 240 
tests for, 241 
Acetone in the blood, 50 

in the gastric contents, 244 
in the urine, 527 
quantitative estimation of, 531 
tests for, 529 
Acetonemia, 50 
Acetonuria, 527 
Acholic stools, 258, 262, 267 
Acids, fatty, in the feces, 319 

organic, in the gastric contents, 240 
Actinomyces hominis, 202, 351 
Actinomycosis, 351 
Adenin in the urine, 427, 585 
Agar. See Culture media. 
Agglutination test, 160 
Agglutinins, 160 
Albumin, acetosoluble, 454 
in the feces, 269, 323 
in the urine, 440 
quantitative estimation of, 467 
residual, in feces, 269 
special test for serum albumin, 466 

for serum globulin, 469 
tests for, 461 
boiling, 464 
nitric acid, 462 
picric acid, 466 
potassium ferrocyanide, 465 
Spiegler's, 466 
trichloracetic acid, 465 
Albuminimeter, 467 
Albuminous expectoration, 358 
Albuminuria, 440 
accidental, 453 
colliquative, 448 
constitutional, 444 
cyclic, 443 
digestive, 452 
febrile, 446 
functional, 442 
hematogenous, 444, 450 
in organic diseases of the kidnevs, 

445 
intermittent, 442 



Albuminuria, mixed, 453 

neurotic, 451 

orthostatic, 443 

physiological, 441 

postural, 443 

referable to circulatory disturb- 
ances, 449 
to impeded outflow of urine, 
450 

renal, 445 

toxic, 451 

transitory, 442 
Albumoses in the blood. 40 

in the feces, 323 

in the gastric contents, 232 

in the urine, 454 

tests for, 469 
Albumosuria, 454 

digestive, 455 

enterogenic, 454 

hematogenic, 455 

hepatogenic, 454 

histogenic, 455 

mixed, 455 

pyogenic, 454 

renal, 455 

A r esical, 455 
Alimentary detritus in feces, 261, 266 
Alkalimeter, Engel's, 21 
Alkaline stools, 316 

urine, 369 
Alkalinity of the blood, 20 
estimation of, 21 
Alkapton in the urine, 518 
Alkaptonuria, 518 
Alloxur bases in the urine, 418, 427 

estimation of, 427 
Almen's solution, 484 
Aloin test for occult blood, 260 
Alveolar epithelium, 336 
Ammo-acids in the urine, 538 
Ammonia in the blood, 43 

in the gastric contents, 242 

in the urine, 415 

estimation of, 416 
Ammoniacal fermentation, 369 
Ammonio magnesium phosphate, 560 
Ammonium urate, 560 
Amebas in the urine, 586 
Amoeba coli, 286 



Q66 



INDEX 



Amoeba coli in the feces, 286 
in the sputum, 339 
Amcebina in the feces, 286 
Amphistomum hominis, 304 
Amphoteric urine, 369 
Amyloid corpuscles in the semen, 624 
Anachlorhydria, 218 
Anacidity, hysterical, 218 
Anadeny of the stomach, 218, 244 
Anemic degeneration of the red cor- 
puscles, 60 
Anguillula aceti, 586 

intestinalis, 312 

stercoralis, 312 
Anguilluliasis, 196 
Aniline dyes, classification of, 125 

-water, gentian violet, 346 
Animal parasites in the blood, 177 
in the feces, 285 
in the sputum, 339 
in the urine, 586 
Anisocytosis, 52 
Anisohypercytosis, 101 
Anisohypocytosis, 101 
Anisonormocytosis, 101 
Ankylostomiasis, 307 
Ankylostomum duodenale, 307 
Annelides, 305 
Anthracosis of the lungs, 357 
Anthrax, bacillus of, 172 
Apiosoma of typhus fever, 190 
Arabinose in urine, 496 
Arneth's karyomorphism, 76, 81 
Arnold's test for diacetic acid, 533 
Ascarides in the feces, 305 

in the urine, 586 
Ascaris lumbricoides, 305 

maritima, 305 

mystax, 305 

Texana, 307 
Asiatic cholera, bacillus of, 283 
Aspergillus fumigatus, 353 
Asthma, bronchial, Charcot-Leyden 

crystals in, 333 
Azoospermatism, 625 
Azurophilic granulation, 72 



Bacillus acidophilus, 280 
coli communis, 282 
dysenterise, 276 
lactis aerogenes, 283 
of anthrax, 172 
of cholera Asiatica, 283 
of diphtheria, 205 
of dysentery, 276 
of Finkler and Prior, 285 
of glanders, 174 
of influenza, 349 
of leprosy, 345 
of Moro, 280 



Bacillus of Oppler and Boas, 250 

of paratyphoid fever, in the blood, 
165 
in the feces, 280 
of plague, 175, 351 
of Shiga, 276 
of smegma, 351 

of tuberculosis, in the blood, 173 
in the feces, 285 
in the gastric contents, 249 
in the meningeal fluid, 616 
in the milk, 634 
in the mouth, 202 
in the nasal discharge, 325 
in the sputum, 344 
in the urine, 580 
methods of staining, 345 
of typhoid fever, in the blood, 159 
in the feces, 278 
in the sputum, 351 
in the urine, 584 
of whooping-cough, 350 
pestis, 175 
pyocyaneus, 282 
smegma, 351, 583 
vulgaris, 281 
Bacteria in blood, 158 
in exudates, 595 
in feces, 273 
in gastric contents, 250 
in milk, 634 
in mouth, 202 
in nasal secretion, 325 
in pus, 600 
in saliva, 200 
in sputum, 344 
in urine, 580 
in vagina, 627 
Bacterial decomposition of the urine, 

369 
Bacteriemia, 158 
colon, 171 
gonococcus, 171 
meningococcus, 171 
paracolon, 165, 171 
paratyphoid, 165 
pneumonia, 166, 170 
proteus, 170 
pyocyaneus, 171 
pyogenic, 168 
staphylococcus, 168 
streptococcus, 169 
typhoid, 159 
Bacteriuria, 580 

idiopathic, 585 
Bacterium lactis aerogenes, 283 

proteus, 170 
Balantidium coli, 293 
Bang's test for albumoses, 470 

for urobilin, 470 
Barfoed's reagent, 233 
Barfurth's reagent, 137 
Basic phosphate of magnesium, 558, 559 



INDEX 



667 



Basophilic leukocytes in the blood, 78 
in the sputum, 334 
perinuclear granules, 76 
Baumann and v. Udranszky's method of 

isolating diamins, 543 
Baumann's test for homogentisinic acid, 

521 
Beckmann's apparatus, 156 
Bence Jones' albumin, 456 
tests for, 471 
Benzoic acid in the urine, 431 
Bile pigment in the blood, 49 
in the feces, 321 
in the gastric contents, 246 
in the urine, 510 
tests for, 512 

Gmelin's, 512 
Huppert's, 512 
Rosenbach's, 512 
Smith's, 512 
Bilharzia hematobia, 195 

eggs in the urine, 586 
Bilharziasis, 195 
Biliary acids in the blood, 50 
in the feces, 320 
in the urine, 512 
tests for, 320 
concretions, 263 
Bilirubin, 510 
Biuret test, 407 
Blastomycetes, 353 
Blood, 17 

acetone in, 50 
albumins in, 39 
albumoses in, 40 
alkalinity of, 21 
ammonia in, 43 
bacteriology of, 158 
biliary constituents in, 49 
carbohydrates in, 40 
chemical examination of, 25 
coagulation of, 27 
color of, 17 
color index, 53 
corpuscles, red, 51 

anemic degeneration of, 60 
behavior toward aniline dyes, 

60 
color index, 53 
crenation of, 53 
granular degeneration of, 61 
enumeration of, 137 
money-roll formation, 53 
nucleated, 65 

osmotic resistance of, 157 
polychromatophilia of, 60 
ring bodies in, 64 
variations in color, 53 
in form, 52 
in number, 54 
in size, 51 
counting, 137 
crisis, 66 



Blood, drying and staining of, 123, 125 

dust, 120 

examination, technique of, 119 

fat in, 45 

fatty acids in, 45 

fibrin in, 39 

gases in, 26 

general characteristics of, 17 
chemistry of, 25 

glycogen in, 42 

hemokonia of, 120 

in the feces, 259, 271 

in the gastric contents, 247 

in the sputum, 328, 335 

in the urine, 508, 567 

iron, 153 

kryoscopy of, 154 

lactic acid in, 48 

leukocytes of, 69 

medicolegal test for, 36 

methods of staining, 125 

microscopic examination of, 51 

mount, 121 

nucleated corpuscles in, 65 

occult, in feces, 260 

odor of, 18 

parasitology of, 177 

peptone in, 40 

pigments of, 29 

plaques of, 118 

plates, 118 

proteids in, 39 

protozoa in, 177 

reaction of, 20 

red corpuscles, 51 

shadows, 568 

specific gravity of, 18 

staining of, 125 

sugar in, 40 

tests for, 36, 260 
aloin test, 260 
Donogany's, 247, 474 
guaiacum, 261 
Heller's, 473 
Miiller and Weber's, 247 

uric acid in, 43 

volume index, 147 

white. See Leukocytes. 

xanthin bases in, 44 
Boas' bulbed stomach tube, 211 

method for estimating lactic acid, 
238 

test for hydrochloric acid, 221 
for lactic acid, 237, 240 
meal, 210 
Boas-Oppler bacillus, 250 
Bogg's method of estimating proteids 

in milk, 637 
Boiling test for albumin, 464 
Bothriocephalus latus, 300 
Bottcher's crystals, 623 
Bottger's test for sugar, 484 
Bremer's diabetic blood test, 61 



668 



INDEX 



Brick-dust sediments, 549 
Brodie and Russell's method of enu- 
merating the plaques, 144 
Browning's spectroscope, 23 
Bubonic plague, bacillus of, 175 
Buccal secretion {see Saliva), 198 
Buerger's capsule stain, 348 
Butyric acid fermentation, 241 

in the feces, 319 

in the gastric contents, 241 
test for, 241 



Cabot's ring bodies, 64 

Cadaverin, 543 

Cahn-Mehring's method of estimating 

fatty acids, 242 
Calcium carbonate, crystals of, 560 
oxalate, crystals of, 357, 551 
phosphate, crystals of, 552 
sulphate, crystals of, 553 
Carbohydrates, digestion of, 233 
in the blood, 40 
in the feces, 323 
in the urine, 474 
tests for, 233 
Carbol fuchsin, 345 
Carbolic acid, estimation of, 517 

test for, 318, 517 
Carbolochloride of iron test for lactic 

acid, 235 
Carbon dioxide hemoglobin, 35 

monoxide hemoglobin, 34 
Casein, in the milk, 637 

test for, Leiner's, 269 
Casts, blood, 572 

classification of, 571 
epithelial, 572 
examination of, 571 
fatty, 574 
fibrinous, 330 
formation of, 570 
granular, 574 
hyaline, 572 
pus, 565 

significance of, 577 
staining of, 572 
urinary, 570 
waxy, 574 
Cellulose in the blood, 42 
Cenomonadina, 289 
Cercomonas intestinalis, 290 
Cerebrospinal fluid, 609 
amount of, 610 
appearance of, 610 
bacteriology of, • 616 
chemical composition of, 612 
cholin in, 613 
cytodiagnosis of, 615 
microscopic examination of, 
615 



Cerebrospinal fluid, reaction of, 612 
specific gravity of, 610 
toxicity of, 618 
Cestodes,. 295 
Chalicosis, 358 

Charcot-Leyden crystals, in the feces, 
272 
in the nasal discharge, 325 
in the sputum, 333 
Cheesy particles in sputum, 330 
Chemical examination of blood, 25 
of cystic fluids, 619 
of exudates, 597 
of feces, 316 
of gastric juice, 214 
of milk, 636 
of pus, 598 
of saliva, 198 
of semen, 624 
of sputum, 358 
of transudates, 590 
of urine, 373 
Chlorides in the urine, 374 
estimation of, 377 

according to Salkowski 

and Volhard, 377 
direct method. 381 
test for, 377 
Cholemia, 49 
Cholera Asiatica, bacillus of, 283 

infantum, bacillus of, 276 
Cholesterin in the blood, 47 
in the feces, 272, 319 
in the sputum, 356 
in the urine, 513 
isolation of, from the feces, 319 
test for, 319 
Cholin in the blood, 50 
Choluria, 510 
Chorion villi, 631 
Chromogens in the urine, 501 
Chyluria, 539 
Chymosin, 230 

estimation of, 232 
test for, 231 
Chymosinogen, 230 
estimation of, 232 
test for, 231 
Ciliata, 293 

Cladothrix asteroidea, 352 
Coagulation of the blood, 27 
Coagulometer, Wright's, 28 
Coating of the tongue, 203 
Coffin4id crystals, 560 
Colloid concretions in ovarian cysts, 

620 
Color index of the blood, 53 
Colostrum, 632 
Comma bacillus, 283 
Concretions, biliary, 263 
fecal, 264 
intestinal, 263 
pulmonary, 333 



INDEX 



669 



Congo-red test for free acids, 219 
Conjugate glucuronates, 497 

sulphates, 392 
Coproliths, 264 
Corpora amylacea, 624 
Crenation of red corpuscles, 53 
Crotonic acid, 535 

Crystals, ammoniomagnesium phos- 
phate, 560 

ammonium urate, 560 

bilirubin, 557 

calcium carbonate, 560 
oxalate, 551 
phosphate, 552 
sulphate, 553 

Charcot-Leyden, 354 

cholesterin, 356 

cystin, 553 

fatty acids, 365 

hematoidin, 557 

hemin, 36 

hippuric acid, 552 

indigo, 561 

in the feces, 271 

leucin, 554 

leukocytic, 354 

magnesium phosphate, 558, 559 

monocalcium phosphate, 552 

neutral calcium phosphate, 558, 
559 

phenylglucosazone, 486 

phosphate of spermin, 621 

Teichmann, 36 

triple phosphate, 560 

tyrosin, 554 

urate of ammonium, 560 

uric acid, 549 

xanthin, 556 
Culture media (preparation of), 

649 
Cursehmann's spirals, 332 
Cylinders, mucous, in the feces, 
262 
in the urine, 577 

urinary, 571 
Cylindroids, 576 
Cylindruria, 571 
Cystein, 396 

Cysticercus cellulosse, 297 
Cystin, 397, 553 
Cvstinuria, 397, 553 
Cysts, colloid, 620 

contents of, 619 

dermoid, 620 

fibrocystic, 621 

hydatid, 621 

ovarian, 619, 621 

pancreatic, 622 

parovarian, 621 
Cytodiagnosis in cerebrospinal fluid, 
615 

in effusions, 592 
Czaplewsky's carbol fuchsin, 345 



Daland's hematokrit, 144 
Dare's hemo-alk*alimeter, 22 

hemoglobinometer, 148 
method of estimating the alka- 
linity of the blood, 22 
Decidual cells, 631 

Decinormal alkali, preparation of, 216 
Dehemoglobinizing method, 177 
Dennige's test for acetone, 530 
Dermoid cysts, 620 
Dextrin in the urine, 496 
Dextrose in the urine. See Glucose. 
Diabetes, 481 

alternans, 423 

Bremer's blood test in, 61 

hepatogenic, 482 

Hirschf eld's form of, 483 

insipidus, 377, 498 

myogenic, 482 

phosphatic, 384 

Williamson's blood test in, 42 
Diabetic chromatophilia, 61 
Diacetic acid in the urine, 532 

tests for, 532 
Diaceturia, 532 
Diamins in the feces, 323 

in the urine, 543 

isolation of, 543 
Diathesis, oxalic acid, 438 

uric acid, 421 
Diazo reaction (see Ehrlich's reaction), 

522 
Dibothriocephalus latus, 300 
Differential density method of estimat- 
ing sugar, 491 

leukocyte count, 143 
Digestion, products of, 232 
Dimethylaminoazobenzol test, 219 
Diphtheria, 205 

Diplococcus meningitidis intracellu- 
laris, 616 

pneumoniae, 348 

in the blood, 166 
Dipylidium caninum, 299 
Distoma Buskii, 304 

capense, 195 

conjunctum, 304 

hematobium, 195, 343 

hepaticum, 302 

heterophyes, 305 

lanceolatum, 303 

pulmonale, 343 

rhatonisi, 304 

sibiricum, 304 

spatulatum, 304 
Distomiasis, 195 
Donne's pus test, 564 
Donogany's blood test, 247, 474 
Doremus' ureometer, 411 
Drigalsky-Conradi medium, 278 
Drysdale's corpuscles, 621 



670 



INDEX 



Dunlop's method of emati sting oxalic 

acid, 439 
Dust particles of Muller, 120 
Dyes, 125 

neutral, 127 

polychrome, 127 
Dysmenorrhea exfoliativa, 630 



Earthy phosphates, 382 
Eberth's bacillus, 278 
Echinococcus, 339 

membranes in the sputum, 333 

in the urine, 587 
polymorphus, 339 
Egg-yellow reaction, 525 
Ehrlich's diazo reaction, 522 

dimethylaminobenzaldehyde reac- 
tion, 526 
egg-yellow reaction, 525 
hemoglobinemic Innenkorper, 65 
triacid stain, 130 
Einhorn's saccharimeter, 492 
Elastic tissue in the sputum, 330, 337 

stain for, 339 
Endocarditis, bacteriology of, 171 
Engel's alkalimeter, 21 

method of estimating the alkalinity 
of the blood, 21 
Entamoeba coli, 289 
dysenterise, 286 
histolytica, 286 

in the feces, 286 
in the sputum, 339 
Enterogenic albumosuria, 454 
Enteroliths, 264 
Eosinate of methylene blue, staining 

with, 129 
Eosinophilia, 102 
Eosinophilic leukocytes in the blood, 77 

in the sputum, 334 
Epithelial cells, alveolar, 336 
ciliated, 336 

in the buccal secretion, 200 
in the feces, 270 
in the sputum, 336 
in the urine, 561 
in the vaginal secretion, 627 
Erythroblasts, 65 
Erythrodextrin, 233 

test for, 233 
Esbach's albuminimeter, 467 

method of estimating albumin, 467 
reagent, 467 
Escherich's stain, 281 
Ethyl sulphide, 397 
Euchlorhydria, 218 
Eustrongylus gigas, 586 
Ewald and Siever's salol test, 253. 
Ewald's test breakfast, 210 
Exudates, 591 



Exudates, bacteriological examination 
of, 595 
chemistry of, 597 
chyloid, 604 
chylous, 604 
cytodiagnosis of, 592 

technique, 594 
hemorrhagic, 591 
in cancer, 594 
in tuberculosis, 592 
inoscopy, 596 
purulent (see Pus), 598 
putrid, 604 
serous, 591 



F arrant' s solution, 571 
Fat in the blood, 45 

estimation of, 46 
in the feces, 262 
in the milk, estimation, 637 
in the urine, 539, 557 
Fatty acids, estimation of, 242 

formation of, 240 

in pus, 602 

in the blood, 45 

estimation of, 47 

in the feces, 319 

in the gastric contents, 240 

in the sputum, 356 

in the urine, 537 

estimation of, 537 

tests for, 241 
casts, 574 
Febrile acetonuria, 528 
albuminuria, 446 
urobilin, 513 
Fecal vomiting, 248 
Feces, 256 

albumin in, 323 
albumoses in, 323 
alimentary detritus in, 261 
amount of, 256 
animal parasites in, 285 
annelides in, 305 
bacteriology of, 273 
biliary acids in, 320 

concretions in, 263 

pigments in, 231 
blood in, 259, 271 
cestodes in, 295 
chemistry of, 316 
cholesterin in, 319 
color of, 258 
composition of, 317 
concretions in, 263 
consistence of, 257 
coproliths in, 264 
crystals in, 271 
enteroliths in, 263 
epithelial cells in, 270 



INDEX 



671 



Feces, fatty acids in, 319 

flagellata in, 289 

foreign bodies in, 265 

form of, 257 

gases in, 317 

hematoporphyrin in, 322 

indol in, 318 

intestinal concretions in, 264 
sand in, 264 

leukocytes in, 271 

macroscopic constituents of, 261 

microscopic constituents of, 265 

mucus in, 262, 273, 322 

number of stools, 256 

odor of, 258 

parasites, animal, 285 
vegetable, 273 

phenol in, 318 

pigments in, 320 

protozoa in, 286 

ptomains in, 323 

purin bodies in, 322 

reaction of, 316 

residual albumin in, 269 

skatol in, 318 

technique in examination of, 265 

trematodes in, 302 

vermes in, 305 
Fehling's method of estimating sugar, 
489 

solution, 484 

test for sugar, 484 
Ferment, milk-curdling, 230 

of saliva, 199 
Fermentation test for sugar, 485 

Schmidt's fecal, 268 
Ferments in the gastric juice, 227 

in the urine, 540 
Ferrocyanide test for albumin, 465 
Ferrometer, Jolles', 153 
Fibrin, 39 

estimation of, 39 

ferment, 27 

in the blood, 39 

in the urine, 459 

test for, 474 
Fibrinogen, 27 
Fibrinous casts, 330 

coagula in the sputum, 330 

in the urine. See Chyluria. 
Ficker's diagnosticum, 164 
Filaria Bancrofti, 191 

demarquai ,191 

diurna, 191 

Mansoni, 191 

nocturna, 191 

ozzardi, 191 

perstans, 191 

sanguinis hominis, 191 

Wuchereri, 191 
Filariasis, 191 
Finkler-Prior bacillus, 285 
Fixation, 124 



Flagellata, 289 
Fleischl's hemometer, 150 
Florence's test for semen, 626 
Folin's method of estimating the acidity 
of the urine, 371 

ammonia, 416 

kreatinin, 434 

sulphates, 394, 395 

urea, 412 

uric acid, 424 
Foreign bodies in the feces, 265 
in the sputum, 333 
in the urine, 587 
Frommer's test, 530 
Furfurol test for bile acids, 320 
Fusiform bacilli of Vincent, 204 



Gabett's staining method, 345 
Galacturia, 539 
Gallstones in the feces, 263 
Garrod's test for hematoporphyrin in 
the urine, 509 
for homogentisinic acid, 520 
Gases in the blood, 26 
in the feces, 317 
in the gastric contents, 242 
in the urine, 541 
Gastric contents, examination of {see 
Gastric juice), 209 
products of digestion, 232 
analysis of, 232 
juice, 209 

acetic acid in, 241 

acetone in, 244 

acidity of, 214 

amount of, 213 

blood in, 247 

butyric acid in, 241 

cause of acidity of, 214 

chemical composition of, 214 

examination of, 214 
chymosin in, 230 
chymosinogen in, 230 
combined hydrochloric acid 

in, 222 
fatty acids in, 240 
ferments in, 227 
free acM in, 217, 219 
gases in, 242 

general characteristics of, 213 
rrydrochloric acid in, 217 
hyperacidity of, 218 
hypersecretion of, 218 
lactic acid in, 233 
methods of obtaining, 211 
microscopic examination of, 

249 
milk-curdling ferment of, 230 
organic acids in, 240 
pepsin in, 227 



672 



INDEX 



Gastric juice, pepsinogen in, 227 
rennin, 230 
secretion of, 209 
zymogens in, 227 
Gastrosuccorrhea mucosa, 246 
Gelatin (see Culture media), 649 
Gerhardt's test for diacetic acid, 532 

for urobilin, 515 
Gerrard and Allan's estimation of sugar, 

490 
Giemsa's stain, 136 
Gigantoblasts (see Megaloblasts), 67 
Glanders, bacillus of, 174 
Glandular fever, 208 
Glaser's method of estimating neutral 

sulphur, 398 
Glucose, 474 

in the blood, 40 

estimation of, 40 

in the urine, 474 

quantitative estimation of, 489 

tests for, 483 
Glucosuria, 474 

digestive, 474 

e saccharo, 474 

ex amylo, 478 

persistent, 481 

transitory, 479 
Glucosuric acid. See Alkapton. 
Glucuronic acid in the blood, 40 

in the urine, 497 
Glycogen in the blood, 42 
test for, 137 
Gmelin's reaction, 512 
Goldhorn's stain, 137 
Gonococcus in the blood, 171 

in the mouth, 203 

in the urine, 585 

in urethral discharge, 603 

Neisser's, 603 

staining of, 585 
Gonorrheal pus, 602, 630 

stomatitis, 202 

threads in the urine, 566 
Gowers' hemoglobinometer, 152 
Gram's method of staining, 205 
Granular degeneration, 61 

casts, 574 
Granulocytes, 69 
Grape sugar. See Glucose. 
Gregarina, 294 

Guaiacum test for blood, 261 
Guanin in the urine, 427 
Gunning's mixture, 413 

test, 530 
Giinzburg's packages, 254 

reagent, 220 



Halitus sanguinis, 18 
Hammerschlag's method of determining 
the specific gravity of blood, 18 



Hammerschlag's method of estimating 

pepsin, 229 
Hastings' stain, 132 
Heart-disease cells, 337 
Heller's test for albumin, 462 

for blood, 473 
Hematemesis, 248 
Hematin, 35 
Hematinuria, 458, 508 
Hematoblasts, 118 
Hematoidin in the blood, 38 
in the sputum, 356 
in the urine, 557 
Hematokrit, 144 

Hematoporphyrin in the blood, 38 
in the feces, 322* 
in the urine, 508 
tests for, 509 
Hematoporphyrinuria, 508 
Hematuria, 508, 567 
Hemin (see Teichmann's crystals), 36 
Hemo-alkalimeter, Dare's, 22 
Hemochromogen, 29 
Hemocytometer of Thoma-Simon, 137 
Hemoglobin, 29 

carbon dioxide, 34 
monoxide, 34 
estimation of, with Dare's instru- 
ment, 148 
with Fleischl's hemometer, 150 
with Gowers' hemoglobinome- 
ter, 152 
with Miescher's instrument, 

152 
with Sahli's instrument, 152 
with Talquist's method, 152 
nitric oxide, 35 

sulphohemoglobin, 35 
tests for, 38, 458 
Hemoglobinemia, 38 
Hemoglobinometer, 147 
Hemoglobinuria, 458 

tests for, 473 
Hemokonia, 120 
Hemometers, 147 
Hemospermia, 625 
Hepatogenic icterus, -511 
Heteroxanthin in the urine, 427 
Hippuric acid in the urine, 429, 
estimation of, 431 
properties of, 430 
test for, 431 
Histon in the urine, 461 

test for, 474 
Hoffmann's test for tyrosin, 556 
Hofmeister's method of estimating hip- 
puric acid, 432 
test for leucin, 556 
Homogentisinic acid in the blood, 49 
in the urine, 518 

estimation of, 520 
isolation of, 520 
Hopkins' method of estimating uric 
acid. See Folin's method. 



INDEX 



673 



Huppert's test for bile pigment, 512 
Hydatid cysts, 339, 621 

echinococcus membranes and 

hooklets in, 339, 621 
sodium chloride in, 621 
succinic acid in, 621 
disease, 339 
Hydrobilirubin, 320 
Hydrocele agar. See Culture media, 
fluid, 50 

cholesterin in, 590 
Hydrochloric acid in the gastric juice, 
214 
amount of, 217 
combined, 222 
deficit, estimation of, 223 
estimation of, according to 
Leo, 226 
according to Martius and 

Luttke, 224 
according to Sahli, 223 
according to Topfer, 222 
free, 219 
tests for, 219 
Hydrogen sulphide, in the gastric con- 
tents, 242 
tests for, 243 
in the urine, 541 
Hydronephrosis, 622 
Hydrothionuria, 541 
Hypalbuminosis, 39 
Hypeosinophilia, 108 
Hyperalbuminosis, 39 
Hyperchlorhydria, 218 
Hyperchromemia, 31 
Hyperinosis, 39 
Hyperleukocytosis, 85 

polynuclear eosinophilic, 102 
neutrophilic, 85 
Hypersecretio acida et continua, 216, 

218 
Hypersecretion, 214, 218 
Hypinosis, 39 

Hypobromite method of estimating 

urea, 409 

solution, 409 

Hypochlorhydria, 218 

Hypoleukocytosis, 85 

polynuclear eosinophilic, 108 
neutrophilic, 97 
Hypoxanthin in the urine, 427 



Icterus, 511 

hematogenic, 511 
hepatogenic, 511 
neonatorum, 511 
urobilin, 514 
Idiopathic bacteriuria, 585 
Ilasvay's reagent, 199 
Indican in the urine, 501 
43 



Indican in the urine, estimation of, 504 

tests for, 503 
Indicanuria, 501 

Indigo-bnie in the urine, 521, 561 
Indigosuria, 521, 561 
Indol in the feces, 318 
tests for, 319 
Indoxyl, 501 

sulphate {see Indican), 501 
Influenza, bacillus of, 174, 349 
Inoscopy, 596 
Inosit in the urine, 498 
Intermittent albuminuria, 442 
Intestinal concretions, 263 

sand, 264 
Iodoform test for lactic acid, 237 
Iodophilia, 83 

demonstration of, 137 
Iodospermin, 626 
Iron in blood, 153 
Irritation forms, 82 
Isohypercytosis, 101 
Isohypocytosis, 101 
Isomastigoda, 289 
Isonormocvtosis, 101 



Jaffe's test for indican, .504 
Jaundice (see Icterus), 511 
Jenner's stain, 129 
Jolles' ferrometer, 153 
Justus' syphilitic blood test, 32 



K 



Kala-azar, Leishmania-Donovani in, 

190 
Karyomorphism, neutrophilic, 101 
Kelling's test for lactic acid, 235 
Kernschatten, 75 
Kjeldahl's method, 413 
Koplik's bacillus, 350 
Krabbea grandis, 302 
Kreatin, 432 

properties of, 433 
Kreatinin, 432 

estimation of, 434 

properties of, 433 

test for, 434 

-zinc chloride, 434 
Kryoscopy of the blood, 154 

of the urine, 546 



Lactic acid 233 

clinical significance of, 233 
estimation of, 238 
fermentation, 235 
in the blood, 48 



674 



INDEX 



Lactic acid, in the gastric contents, 233 
in the urine, 535 
mode of formation, 233 
tests for, 235 
Boas', 237 
Kelling's, 235 
Strauss', 235 
Uffelmann's, 235 
Vournaso's, 236 
Lactodensimeter of Quevenne, 635 
Lactose in the urine, 495 
in the milk, 637 
Laiose in the urine, 495 
Large mononuclear leukocytes, 73 

clinical variations of, 112 
Laveran's organism. See Malarial organ- 
ism. 
Legal's test for acetone, 529 
Leiner's test for casein, 269 
Lei hmania-Donovani, 190 
Leishman's stain, 134 
Leo's method of estimating hydrochloric 

acid, 226 
Leprosy, bacillus of, 174 
Leptothrix buccalis, 204 
Leube's test of motor power of stomach, 

252 
Leucin, 554 
Leucopenia, 97 
Leukocytes, 69 
basophilic, 78 
degenerative changes, 75 
differential enumeration, 143 
differentiation according to their 
behavior toward aniline dyes, 70 
enumeration of, 140 
eosinophilic, 77 

estimation of the number of, 137 
general differentiation of the vari- 
ous forms, 69 
in the blood 69 
in the exudates, 592 
in the feces, 271 
in the sputum, 334 
in the urine, 563 
irritation forms, 82 
large mononuclear, 73 
lymphocytes, 70 
mast-cells, 78 
myelocytes, 80 
neutrophilic, 74 
oxyphilic, 77 
pigmented, 185 
phlogocytes, 82 
polymorphonuclear, 74 
polynuclear, 74 
small mononuclear, 70 
splenocytes, 73 
transition forms, 74 
variations in number of, 84 
Leukocytic crystals, 354 
Leukocytosis (see Hyperleukocytosis), 
84 



Leukemia, lymphatic, 112 

myelogenous, 116 
Levulose in the urine, 495 
Lieben's test for acetone, 529 
Lientery, 261 
Lipacidemia, 46 
Lipaciduria, 537 
Lipase, in the gastric juice, 232 

in the urine, 540 

test for, 540 
Lipemia, 46 
Lipuria, 539, 558 
Lochia, 629 

alba, 629 

rubra, 629 
Loffler's bacillus, 205 

methylene- blue solution, 205 
Lohnstein's saccharimeter, 492 
Lowy's method of estimating the alka- 
linity of the blood, 21 
Lymphocytes, 70 

small, 70 

large, 71 
Lymphocytosis, 73, 109 
Lymphopenia, 73, 112 



M 



Macrocytes, 51 
Macrocythemia, 51 
Macrolymphocytes, 71 
Magnesia, soaps of, in the urine, 557 
Magnesium phosphate, 558, 559 
Malachite green medium of Lentz, 278 
Malaria, plasmodium of, 177 
Malta fever, bacillus of, 174 
Maltose in the urine, 496 
Mammary secretion, 633 
Marsh gas in the gastric contents, 242 
Martius and Liittke's method of esti- 
mating hydrochloric acid, 224 
Masons' lung (see Siderosis), 358 
Mast-cells, 78 

clinical variations of number, 
113 
May-Griinwald stain, 130 
Meconium, 323 

Medicolegal test for blood, 36 
Megaloblasts, 67 
Megalocytes, 51 
Megastoma entericum, in the feces, 292 

in the gastric contents, 251 
Melanin in the urine, 516 

tests for, 516 
Melanogen, 516 
Melanuria, 516 
Membranous dysmenorrhea, vaginal dis 

charge in, 630 
Meningeal fluid, examination of, 609 
Meningococcus, 171, 616 
Menstruation, vaginal discharge in, 629 
Metalbumin in ovarian cysts,* 619 



INDEX 



675 



Metamyelocytes, 76, 81 
Methemoglobin, 37 

sulphide, 35 
Methemoglobinemia, 37 
Methemoglobinuria, 458 
Methane. See Marsh gas. 
Methylene azure, 132 
Mett's method of estimating pepsin, 229 
Microblasts, 69 
Micrococcus catarrhalis, 351 

melitensis, 174 

tetragenus, 351 

urese, 581 

zymogenes, 171 
Microcytes, 51 
Microcythemia, 51 
Microlymphocytes, 70 
Miescher's hemometer, 152 
Milk, 633 

chemical composition of, 633 

cows', 633 

examination of, 636 

fat in, estimation of, 637 

human, 633 

in disease, 634 

lactose, estimation of, 637 

proteids of, estimation of, 637 

secretion of, in the adult female, 
633 
in the newly born, 631 

specific gravity of, 635 

witches', 631 
Milk-curdling ferment in the gastric 

juice, 230 
Millon's reagent, 470 
Money-roll formation of red corpuscles, 

53 
Monocalcium phosphate, 552 
Moro's bacillus, 280 
Motor power of stomach, examination 
of, 252 
Leube's method, 252 
salol test of Ewald and Sievers, 
253 
Mouth, actinomycosis of, 202 

secretions of, 198 

tuberculosis of, 202 
Mucin in the feces, 322 

in the urine, 460 
Mucor corymbifer, 353 
Mucous cylinders in the feces, 262 

in the urine, 577 
Mucus in the feces, 262, 273 

in the gastric contents, 246 
Muller-Weber test for blood, 247 
Murexid test, 424 
Myelemia, 116 

Myelin granules in the sputum, 336 
Myelocytes, amblychromatic, 80 

basophilic, 82 

eosinophilic, 81 

neutrophilic, 76, 80 

trachychromatic, 80 
Myelocytosis, 114 



N 



Nasal catarrh, 325 
secretion, 325 

cerebrospinal fluid in, 325 
characteristics of, 325 
Charcot-Leyden crystals in, 325 
in disease, 325 
Neisser, gonococcus of, 603 
Neisser's stain, 205 
Nematodes, 305 
Nessler's reagent, 250 
Neusser's granules, 76 
Neutral dyes, 127 

phosphate of calcium in the urine, 

558, 559 
sulphur in urine, 396 
Neutrophilic karymorphism, 101 

leukocytes, 74 
Nitric acid test for albumin, 462 

oxide hemoglobin, 35 
Nitrites in the saliva, 199 
Nitrogen in the urine, 401 
estimation of, 413 

according to Kjeldahl, 413 
Nitrogenous equilibrium, 402 
metabolism, 401 
Nitroprusside of sodium, as a test for 

acetone. See Legal's test. 
Normal urobilin, 499 
Normoblasts, 65 
Nose, secretion from, 325 
Nucleated red corpuscles, 65 
Nucleo-albumin in the urine, 460 

test for, 472 
Nucleohiston in the urine, 461 
Nummular sputum, 329 
Nylander's test for sugar, 484 



Obermayer's reagent, 503 
Obermeier, spirochete of, 189 
Occult bleeding, 247 
Oidium albicans, 202 
Oligochromemia, 31 
Oligocythemia, 31, 57 
Oliguria, 365 
Opsonins, 639 

Simon's index, 643 
technique, 642 
Wright's index, 643 
Orcin test for pentoses, 497 
Organized sediments of the urine, 561 
Ott's test, 472 
Ovarian cysts, 619, 621 
Oxalate of calcium crystals in the spu- 
tum, 551 
in the urine, 551 
Oxalic acid, diathesis, 438 
in the urine, 436 
properties of, 438 
quantitative estimation of, 439 



676 



INDEX 



Oxalic acid, tests for, 439 
Oxaluria idiopathica, 438 
Oxaluric acid, 436 
Oxyamygdalic acid, 536 
Oxybutyric acid, j3, in the urine, 533 

estimation of, 533 
Oxyhemoglobin, 29 
Ox3^philic leukocytes, 77 
Oxyuris vermicularis, 307 
Ozena, 325 



Pancreatic cysts, 622 

trypsin in, 622 
juice in the gastric contents, 
246 
Pappenheim's methyl-green pyronin, 

595 
Paragonimus Westermanni, 343 
Paramoeba hominis, 289 
Paramecium coli, 293 
Paramucin, 619 
Parasites in the blood, 177 
in the feces, 285 
in the gastric contents, 249 
in the sputum, 339 
in the urine, 580 
malarial, 177 
Paratyphoid fever, bacillus of, 165 
in the blood, 165 
in the urine, 585 
Paraxanthin in the urine, 427 
Patein's albumin, 454 
test for, 499 
Pentoses in the urine, 496 

tests for, 497 
Pepsin in the gastric juice, 227 
estimation of, 229 
tests for, 229 
Pepsinogen in the gastric juice, 227 
estimation of, 230 
tests for, 229 
Peptones in the blood, 40 
in the urine. 457 
test for, 470 
Peptonuria, 457 
Pertussis bacillus, 350 
Pettenkofer's test, 320 
Pfeiffer-Widal reaction, 160 
Phagocytes, 69 
Phagocytosis, 69, 186, 639 
Pharyngomycosis leptothrica, 203 
Phenol, 517 

estimation of, 517 
in the feces, 318 
in the urine, 517 
tests for, 318, 517 
Phenylcyanate method of isolating 

diamins, 545 
Phenylglucosazone, 486 
Phenylhydrazin test for sugar, 486 
Phlogocytes, 82 



Phloroglucin test for pentoses, 497 

vanillin test for hydrochloric acid, 
220 
Phosphates in the urine, 382 

estimation of, 387 

removal of, from urine, 390 

separate estimation of alkaline 
and earthy, 390 

tests for, 386 
Phosphatic diabetes, 384 

sediments in the urine, 558 
Picric acid test for albumin, 466 
Piria's test for tyrosin, 556 
Plague bacillus, 175, 351 
Plaques, 118 

enumeration of, 144 
Plasma of the blood, 17, 24 
Plasmodium malariae, 178 

crescentic bodies, 181 

flagellate bodies, 183 

gametes, 182 

hyaline bodies, 178 

macrogametes, 184 

merozoites, 180 

microgametes, 184 

microgametocytes, 184 

ookinetes, 184 

ovoid bodies, 181 

pigmented extracellular bodies, 
182 
intracellular bodies, 179 

polymites, 183 

schizogony of, 181 

segmenting bodies, 180 

spherical bodies, 181 

sporogony, 184 

sporozoites, 184 

staining of, 178 
Pneumococcus, 348 
Pneumoconioses, 357 
Pneumonia, diplococcus of, 348 

in the blood, 166 
sputum in, 348 
Pneumonomycosis aspergillina, 354 
Poikilocytes, 52 
Poikilocytosis, 52 
Polarimeter, 494 

Polarimetric test for sugar, 487, 494 
Pole bacillus, 350 
Polychromasia, 60 
Polychromatophilia, 60 
Polycythemia, 56 
Polymastigina, 290 
Polypeptides in the urine, 470 
Polyuria, 363 
Proleukocyte, 81 
Promyelocyte, 80 
Propepsin, 227 
Prostatic fluid, 624 
Proteus vulgaris, 281 
Protozoa, 286 
in pus, 601 
in the blood, 177 



INDEX 



677 



• Protozoa in the feces, 286 

in the gastric contents, 251 

in the sputum, 339 

in the urine, 580 
Pseudocasts, 576 
Pseuclodiphtheria bacillus, 206 
Pseudogonococci, 603 
Pseudomucin, 619 
Ptomains in the feces, 323 

in the urine, 542 
isolation of, 543 
Ptvalin, 199 

test for, 199 
Purin, 418 

bases, in the feces, 322 
in the urine, 427 
Pus, 598 

bacteria in, 600 

casts, 565 

chemistry of, 598 
. corpuscles, in the urine, 567 
enumeration of, 567 

crystals in, 602 

detritus in, 600 

examination of, 602 

general characteristics of, 598 

giant corpuscles in, 600 

gonorrheal, 602 

in the feces, 271 

in the gastric contents, 248 

in the urine, 563 

leukocytes in, 599 

microscopic examination of, 599 

parasites in, 600 

protozoa in, 601 

red corpuscles in, 600 

tests for, 564 
Putrescin, 543 
Pyroplasma hominis, 191 
Pyuria, 563 

Q 

Quevenne's lactodensimeter, 635 



Ray fungus, 351 

Red blood corpuscles, 51 

anemic degeneration of, 61 
behavior toward aniline dves, 

60 
enumeration of, 140 
granular degeneration of, 61 
nucleated forms, 65 
variations in color, 53 
in form, 51 
in number, 54 
in size, 51 
Relapsing fever, spirochete of, 189 
Resorcin test, 221 

Resorptive power of the stomach, ex- 
amination of, 253 



Rejmold's test for acetone, 529 

Rhamnose in urine, 496 

Rhizopoda, 286 

Riegel's test dinner, 210 

Ring bodies of Cabot, 64 

Romano wsky's method of staining, 132 

Rosenbach's reaction, 506 

test for bile pigments, 512 
Ross' dehemoglobinizing method, 177 
Round-worms, 305 



Saccharimeter of Einhorn, 492 
of Lohnstein, 492 
of Soleil-Ventzke, 494 
Saccharomyces cerevisise. See Yeast. 
Sahli's desmoid reaction, 255 
estimation of free HC1, 223 
hemoglobinometer, 152 
Saliva, 198 

chemistry of, 198 
general characteristics of, 198 
in special diseases of the mouth, 202 
in the gastric contents, 246 
microscopic examination of, 199 
nitrites in, 199 

pathological alterations of, 201 
ptyalin in, 199 
test for nitrites, 199 
for ptyalin, 199 
for sulphocyanides, 199 
Salivation, 201 

Salkowski's method of estimating oxalic 
acid in urine, 440 
xanthin bases in urine, 427 
test for albumoses, 469 
for phenol, 517 
Salkowski-Volhard method of estimat- 
ing the chlorides in urine, 377 
Salol test of Ewald and Sievers, 253 
Salzer's test meal, 210 
Sand, intestinal, 264 
Sarcina pulmonalis, 354 
urinse, 586 
ventriculi, 250 
Scarlatina, pharyngeal secretion in, 208 
Schaumer'smethylene-blue-pyronin,206 
Scherer's test for leucin, 556 
Schizomycetes in the feces, 276 
Schmalz and Peiper's method of deter- 
mining the specific gravity of the 
blood, 19 
Schmidt's fecal fermentation test, 268 
Schuffner's stippling, 64 
Sediments in acid urines, 546 
in alkaline urines, 558 
urinary, 546 

ammoniomagnesium phos- 
phate in, 560 
ammonium urate in, 560 
amorphous urates in, 550 



678 



INDEX 



Sediments, urinary, bacteria in, 580 

basic magnesium phosphate in, 

558, 559 
bilirubin in, 557 
blood corpuscles, red, 567 
brick-dust, 549 
calcium carbonate in, 560 
oxalate in, 551 
sulphate in, 553 
cylindroids, 576 
cystin in, 553 
epithelial cells in, 561 
fat in, 557 

foreign bodies in, 630 
hematoidin in, 557 
hippuric acid in, 552 
indigo in, 561 
leucin in, 554 
leukocytes in, 563 
mode of examination of, 548, 

571 
monocalcium phosphate in, 

552 
mucous cylinders, 577 
neutral calcium phosphate in, 

559 
non-organized, 549 
organized, 561 
parasites in, animal, 586 

vegetable, 580 
protozoa in, 586 
soaps of lime and magnesium 

in, 557 
spermatozoa in, 579 
tube casts in, 570 

tumor particles in, 587 
tyrosin in, 554 
urates in, 550, 560 
uric acid in, 549 
xanthin in, 556 
Semen, 624 

chemistry of, 624 
general characteristics of, 624 
microscopic examination of, 624 
pathology of, 624 
recognition of, in stains, 625 
spermatic crystals in, 624 
spermatozoa in, 624 
Sepsis, organisms in the blood in, 158 
Septic factor, Simon's, 90, 108 
Serosamucin, 597 
Serous exudates, 591 
Serum albumin in the blood, 28 
in the urine, 441 

estimation of, 467 
tests for, 466 
globulin in the blood, 28 
in the urine, 454 

estimation of 469 
test for, 469 
Shiga's bacillus, 276 
Siderosis, 358 
Simon's counting chamber, 137, 139 



Simon's opsonic index, 643 
septic factor, 90, 108 
ureometer, 409 
Skatol in the feces, 318 

tests for, 319 
Sleeping sickness, organism of, 188 
Slides, 120 

Small mononuclear leukocytes, 70 
Smegma bacillus, 351, 583 
Smith's test for bile pigment, 512 
Soaps of lime and magnesium in the 

urine, 557 
Sodium chloride in hydatid fluid, 621 
Spermatic crystals, 623 
Spermatocystitis, 580 
Spermatorrhea, 580 
Spermatozoa in the semen, 624 

in the urine, 579 
Spermin, 624 
Spiegler's reagent, 466 
Spirals of Curschmann, 332 
Spirilla of Vincent's angina, 204 
Spirochete Obermeieri, 189 
pallida, in blood, 191 

in syphilitic material, 606 
Splenocytes, 73 
Sporozoa, 294 

Spotted fever, organism of, 191 
Sputum, 326 

Amoeba coli in, 339 

amount of, 327 

bacteria in, 344 

blastomycetes in, 353 

blood in, 335 

cheesy particles in, 330 

chemistry of, 358 

color of, 328 

concretions in, 333 

configuration of, 329 

consistence of, 327 

crudum, 329 

crystals in, 354 

Curschmann's spirals in, 332 

Diplococcus pneumoniae in, 348 

Distoma pulmonale in, 343 

echinococcus in, 333 

elastic tissue in, 330, 337 

epithelial cells in, 336 

fibrinous casts in, 330 

foreign bodies in, 333 

general characteristics of, 326 

globosum, 329 

heterogeneous, 329 

homogeneous, 329 

influenza bacillus in, 349 

leukocytes in, 334 

macroscopic constituents of, 330 

microscopic examination of, 334 

nummular, 329 

odor of, 328 

parasites, animal, in, 339 
vegetable, in, 344 

specific gravity of, 329 



INDEX 



679 



Sputum, streptothrices in, 351 
Taenia echinococcus in, 339 
technique in the examination of, 

326 
tubercle bacillus in, 344 
Staining, methods of, 129 

principles of, 125 
Staphylococcus pyogenes albus, 
169 
aureus, 169 
citreus, 169 
Steatorrhea, 262 
Stercobilin, 320, 513 
Stercoraceous material in the vomit, 

248 
Stimulation forms, 82 
Stomach, motor power of, 252 
rate of absorption in, 254 
tube, 211 

contra-indications to its use, 

211 
its introduction, 211 
washing, 212 
Stomatitis, catarrhal, 202 
gonorrheal, 202 
ulcerative, 202 
Stools. See Feces. 
Strauss' test for lactic acid, 236 
Strecker's test for xanthin, 557 
Streptococcus pyogenes, 170 
brevis, 170 
conglomeratus, 170 
longus, 170 
Streptothrices, 351 
Streptothrix actinomycotica, 352 
eppingeri, 352 
hominis, 352 
pseudotuberculosa, 352 
Strongyloides, 308 

intestinalis, 312 
Strongylus duodenalis, 308 
Stycosis, 358 

Succinic acid in hydatid fluid, 621 
Sudan stain for fat, 572 
Sugar in the blood, 40 
in the urine, 474 

estimation of, 489 
tests for, 483 
Sulphanilic acid test. See Ehrlich's 

reaction. 
Sulphates in the urine, 390 

estimation of total, 394 
conjugate, 392 
estimation of, 395 
mineral, 391 
tests for, 394 
Sulphocyanides, in the saliva, 199 

in the urine, 396 
Sulphohemoglobin, 35 
Sulphur, neutral, in urine, 396 
estimation of, 398 
Syphilitic blood test of Justus, 32 
Syphilis. See Spirochete pallida. 



Taenia Africana, 299 

canina, 299 

cucumerina, 299 

diminuta, 299 

echinococcus, 339 

flavopunctata, 299 

lata, 300 

Madagascariensis, 299 

mediocanellata, 295 

nana, 297 

saginata, 295 

solium, 297 
Talquist's hemoglobinometer, 152 
Tartar, 203 

Taurocarbaminic acid in urine, 396 
Teichmann's crystals, 36 
Test breakfast of Boas, 210 

of Ewald and Boas, 210 

dinner of Riegel, 210 

meal of Salzer, 210 

meals, 210 
Thiosulphates in urine, 396 
Thoma-Zeiss' hemocytometer, 140 
Thrush, 202 
Toison's fluid, 140 
Tollen's orcin test, 497 

phloroglucin test, 497 
Tongue, coating of, 203 
Tonsillitis, 204 
Tonsils, coating of, 203 
Topfer's method of estimating hydro- 
chloric acid, 222 

test for hydrochloric acid, 219 
Transition forms, 74 
Transudates, 588 

albumin in, 589 

chemistry of, 590 

coagulation of, 590 

general characteristics of, 588 

microscopic examination of, 591 

specific gravity of, 588 
Trematodes, 302 

Treponema pallidum, in blood, 191 
in syphilitic material, 606 
Triacid stain, Ehrlich's, 130 
Pappenheim's, 126 
Trichina spiralis, 311 
Trichloracetic acid test, 465 
Trichocephalus dispar, 311 
Trichomonads in the feces, 291 

in the sputum, 339 

in the stomach contents, 251 

in the urine, 586 

in vaginal discharges, 628 
Trichomonas vaginalis, 291 
Trichotrachelides, 311 
Triple phosphate crystals in the sputum, 
357 
in the urine, 560 
Tripperfaden, 566 
Trommer's test, 483 



680 



INDEX 



Tropeolin test for hydrochloric acid, 221 
Trypanosoma gambiense, 188 
Trypanosomiasis, 187 
in the blood, 187 
in the cerebrospinal fluid, 618 
Trypsin in pancreatic cysts, 622 

test for, 622 
Tube casts in the urine, 571 
amyloid, 575 
blood, 572 

clinical significance of, 577 
compound hyaline, 573 
epithelial, 572 
fatty, 574 
granular, 574 
hyaline, 572 

mode of examination of, 571 
pseudo-, 576 
pus, 565 
staining of, 572 
true, 572 
waxy, 574 
Tubercle bacillus, 344 

cultivation of, 347 
detection of, 345 
in the blood, 173 
in the cerebrospinal fluid, 616 
in the feces, 285 
in the milk, 634 
in the mouth, 202 
in the sputum, 344 
in the urine, 582 
staining of, 345 
Tuberculosis, bacillus of, 344 
Tumor particles in the gastric contents, 
252 
in the urine, 587 
Tlirck's counting chamber, 140 
Typhoid fever, bacillus of, 159, 278 
in the blood, 159 
in the feces, 278 
in the sputum, 351 
in the urine, 584 
Typhus fever, apiosoma of, 190 
Tyrosin, in the sputum, 357 
in the urine, 538, 554 
test for, 556 



U 



Uffelmann's test for lactic acid, 235 
Ulceromembranous angina of Vincent, 

204 
Uncinaria Americana, 308 

duodenalis, 307 
Unna-Tanzer stain, 339 
Urates in urinary sediments, 550, 560 
Urea in the blood, 42 
in the urine, 399 

estimation of, 409, 412 
origin of, 399 
properties of, 407 
tests for, 407 



Urea nitrate, 408 
oxalate, 408 
Uremia, 43 
Ureometers, 409 
Doremus', 411 
Heinz, 411 
Simon's, 409 
Uric acid, 417 

crystals of, 549 
diathesis, 421 
estimation of, 424 

Folin's method, 424 
Hopkins' method, 424 
L u d w i g-Salkowski me- 
thod, 427 
in blood, 43 
in sediments, 588 
in urine, 417 
properties of, 423 
tests for, 424 
Urine, 360 

acetone in, 527 

acidity of, 371 

albumins in, 440 

albumoses in, 454 

alkapton in, 518 

alloxur bases in, 427, 556 

amino-acids in, 538 

ammonia in, 415 

amount, 362 

animal parasites in, 586 

bacteria in, 580 

Bence Jones' albumin in, 456 

benzoic acid in, 431 

bile acids in, 512 

pigments in, 510 
blood in, 567 
blue, 521 

carbohydrates in, 474 
carbonates in, 560 
casts in, 570 
chemistry of, 373 
chlorides in, 374 
cholesterin in, 513 
chromogens in, 501 
chyle in, 539 
color of, 360 
consistence of, 362 
crotonic acid in, 535 
cystin in, 397, 553 
dextrin in, 496 
diacetic acid in, 532 
Ehrlich's diazo reaction, 522 

benzaldehyde reaction, 526 
epithelium in, 561 
fat in, 539, 557 
fatty acids in, 537 
ferments in, 540 
fibrin in, 459 
foreign bodies in, 587 
gases in, 541 
general appearance of, 360 

chemical composition of, 373 



INDEX 



681 



Urine, glucose in, 474 

glucuronic acid in, 497 

green, 521 

hematoporphyrin in, 508 
\ hemoglobin in, 458 
[ hippuric acid in, 429, 552 
L hist on in, 461 

homogentisinic acid in, 518 

indican in, 501 
[ indigo in, 561 

inosit in, 498 

kreatin in, 432 

kreatinin in, 432 

kryoscopy in, 546 

lactic acid in, 535 
L lactose in, 495 

laiose in, 496 

leucin in, 554 

leukocytes in, 563 

levulose in, 495 

maltose in, 496 

melanin in, 516 

microscopic examination of, 546 

mineral ash, estimation of, 373 

neutral sulphur in, 396 

nitrogen in, 402 

nucleo-albumin in, 460 

nucleohiston in, 461 

odor of, 362 

organized sediments in, 561 

oxalates, 551 

oxalic acid in, 436 

oxaluric acid in, 436 

oxyamygdalic acid, 536 

oxybutyric acid in, 533 

parasites in, 580 

pentoses in, 496 

peptone in, 457 

phenol in, 517 

phosphates in, 382, 552, 558 

pigments in, 498 

referable to drugs in, 521 

ptomains in, 542 

pus in, 563 

quantity of, 362 

reaction of, 369 

sediments in, 546 

serum albumin in, 441 
globulin in, 454 

solids in, 368 

specific gravity of, 366 

spermatozoa in, 579 

sugar in, 474 

sulphates in, 390 

sulphur neutral in, 396 

tube casts in, 570 

tumor particles in, 587 

tyrosin in, 554 

urates in, 550, 560 

urea in, 399 

uric acid in, 417, 549 

urobilin in, 513 

urochrome in, 499 



Urine, uroerythrin in, 500 

urohematin in, 506 

urohematoporphyrin in, 508 

urorosein in, 507 

vegetable parasites in, 580 

xanthin bases in, 427, 556 
Urines, blue, 521 

green, 521 
Urinometers, 367 
Urobilin, febrile, 513 

in the blood, 49 

in the urine, 513 

normal, 499 

pathological, 513 

tests for, 49 

Braunstein's, 515 
Gerhardt's, 515 
Schlesinger's, 515 
spectroscopic, 515 
Urobilinogen, 513 
Urobilinuria, 513 
Urochrome, 499 
Uroerythrin, 500 
Urofuscohematin, 508 
Urohematin, 506 
Urohematoporphyrin, 508 
Urorosein, 507 
Uroroseinogen, 507 
Urorubrohematin, 508 



Vaginal blennorrhea, 628 
discharges, 627 

bacteria in, 627 

during menstruation, 629 

following parturition, 629 

general description of, 627 

in abortion, 631 

in gonorrhea, 630 

in membranous dvsmenorrhea, 
630 

in uterine cancer, 630 

in vaginitis, 629 

in vulvitis, 629 

parasites in, 628 

reaction of, 627 
Vaginitis exfoliativa, 630 
Vaughan's granules, 64 
Vincent's angina, 204 

fusiform bacillus, 204 
Vitalli's test for pus, 564 
Volume index, 147 
Vomited material, 244 

bile in, 246 

blood in, 247 

food material in, 244 

mucus in, 245 

odor of, 249 

pancreatic juice in, 246 

parasites in, 249 

pus in, 248 



682 



INDEX 



Vomited material, saliva in, 246 

stercoraceous material in, 248 
Vomitus matutinus, 246 
Von Fleischl's hemometer, 150 
Vournaso's test for lactic acid, 236 



W 



Wang's estimation of indican, 504 
Waxy casts, 574 
Weigert-Ehrlich stain, 346 
Weigert's elastic-tissue stain, 339 
WeyFs test for kreatinin, 434 
Whetstone crystals. See Uric acid. 
White blood corpuscles. See Leuko- 
cytes. 
Whooping-cough, bacillus of, 350 
Widal's serum test, 160 



Williamson's blood test in diabetes, 42 
Worms. See Vermes. 
Wright's coagulometer, 28 
stain, 135 



Xanthin bases in the blood, 44 

in the feces, 322 

in the urine, 418, 427, 556 
estimation of, 427 
Xylose in urine, 496 



Zappert-Ewing counting chamber, 140 
Ziehl-Neelsen s^ain, 346 
Zymogens in the gastric juice, 227 



j W 15 l%7 



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